Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

Klebsiella oxytoca causes colonization resistance against multidrug-resistant K. pneumoniae in the gut via cooperative carbohydrate competition.

Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

Effects of fructans and probiotics on the inhibition of Klebsiella oxytoca and the production of short-chain fatty acids assessed by NMR spectroscopy.

Generally, the selection of fructans prebiotics and probiotics for the formulation of a symbiotic has been based on arbitrary considerations and in vitro tests that fail to take into account competitiveness and other interactions with autochthonous members of the intestinal microbiota. However, such analyzes may be a valuable step in the development of the symbiotic. The present study, therefore, aims to investigate the effect of lactobacilli strains and fructans (prebiotic compounds) on the growth of the intestinal competitor Klebsiella oxytoca, and to assess the correlation with short-chain fatty acids production. The short-chain fatty acids formed in the fermentation of the probiotic/prebiotic combination were investigated using NMR spectroscopy, and the inhibitory activities were assessed by agar diffusion and co-culture methods. The results showed that Lactobacillus strains can inhibit K. oxytoca, and that this antagonism is influenced by the fructans source and probably associated with organic acid production.

Multidrug-resistant Klebsiella oxytoca ventriculitis, successfully treated with intraventricular tigecycline: A case report.

A 38-year-old male presented to the hospital with headache, fever, and meningeal signs. He had undergone a surgical review of a ventriculoperitoneal shunt system one month earlier. A head computed tomography scan showed hydrocephalus. His medical history included a human immunodeficiency virus infection identified four years before and resolved cryptococcal meningitis, which had necessitated the implantation of the shunt system. Ventricular cerebrospinal fluid (CSF) was obtained, which showed inflammation and, in culture, grew a Gram-negative bacillus identified as multidrug-resistant Klebsiella oxytoca. The shunt was removed and a ventricular drain was installed. Treatment with meropenem and amikacin was established without a response; the CSF white blood cell count continued to increase, with cultures remaining positive. The patient's clinical condition deteriorated to stupor. With informed consent, intraventricular (ITV) treatment with tigecycline was initiated at a dose of 5 mg every 24 h and, three days later, the CSF cultures were negativized. Tigecycline levels in the CSF were quantified by liquid chromatography with ultraviolet detection and showed peak concentrations achieved at two hours after the dose of between 178 and 310 µg/mL. After 11 days of treatment with ITV tigecycline and eight negative CSF cultures, a new CSF shunt was installed. During follow-up review 10 months later, the patient reported he was working. The dose of tigecycline used in this study produced levels 15 to 20 times the minimum inhibitory concentration of the bacteria for up to six hours with adequate tolerance.

Expression of adhesin genes and biofilm formation among Klebsiella oxytoca clinical isolates from patients with antibiotic-associated haemorrhagic colitis.

PURPOSE: Biofilm formation and resistance to last-line antibiotics have restricted chemotherapy options toward infection eradication. METHODOLOGY: Fifty K. oxytoca isolates were collected from patients with antibiotic-associated haemorrhagic colitis (AAHC). Antibiotic susceptibility tests were conducted and phenotypic biofilm formation was assessed using microtitre tissue plate (MTP) assay. PCR was employed to amplify the adhesins, extended-spectrum ß-lactamases (ESBLs), carbapenemase and colistin resistance genes. The expression of adhesin genes was evaluated using quantitative real-time PCR (RT-qPCR).Results/Key findings. The previous antibiotic consumption and hospitalization (P<0.05) and older ages (P=0.0033) were significantly associated with AAHC. None of the isolates produced biofilm strongly, but 70% of them produced moderate-level biofilm. The blaCTX-M (12/14), the blaIMP (8/14 MICIMI =4 µg ml-1 ) and blaOXA-48-like (5/14) and mcr-1 (4/14) genes were predominant, three of which harbouring all the genes. The expression of matB (0.023) and mrkA (0.011) was significantly different between multidrug-resistant and susceptible isolates. Furthermore, moderately biofilm producer isolates significantly exhibited higher expression of fimA (P=.0117), pilQ (P=0.002) and mrkA (P=0.020) genes compared to biofilm non-producers. No significant difference regarding gene expression was observed among ESBL alleles. CONCLUSION: Bacterial attachment by adhesins and biofilm formation among extensive drug-resistant K. oxytoca isolates hinder the efficient infection eradication. Hence, control and surveillance studies should be performed and other therapeutic auspicious approaches must be taken into account against AAHC, biofilm formation and drug resistance spread. Furthermore, previous antibiotic consumption and long-term hospitalization should be controlled.

Pneumatosis intestinalis and hepatic portal venous gas associated with gas-forming bacterial translocation due to postoperative paralytic ileus: A case report.

RATIONALE: Pneumatosis intestinalis (PI) and hepatic portal venous gas (HPVG) are rare but potentially lethal conditions in which gas pathologically accumulates in the portal vein and intestinal wall, respectively. Proposed mechanisms include flatus escaping through an injured intestinal mucosa into the submucosa and thence into the portal venous system, or bacterial translocation (BT) of gas-forming enteric microorganisms from the gut into and through the intestinal wall to other organs. However, there has been no clear histopathological evidence to support these hypotheses. PATIENT CONCERNS: A 61-year-old man underwent sigmoidectomy for colonic adenocarcinoma. Postoperatively, he developed paralytic ileus and then had a sudden cardiopulmonary arrest. DIAGNOSES: PI and HPVG were found at autopsy, presumably caused by the postoperative paralytic ileus and associated with BT of gas-forming organisms. INTERVENTIONS: Cardiopulmonary resuscitation was unsuccessful. OUTCOMES: Postmortem imaging indicated the presence of massive PI and HPVG. At autopsy, there was marked intestinal emphysema with diffuse ischemic mucosal necrosis and severe pneumatosis in the stomach and intestine and marked gaseous dilation of the intrahepatic portal veins. Postmortem bacterial cultures revealed enteric bacteria in the peripheral blood and liver tissue. LESSONS: Postoperative ileus leading to intestinal mucosal damage may be associated with BT of gas-forming enteric bacteria and the rapid onset of PI and HPVG with a lethal outcome.

Polymicrobial infections: Do bacteria behave differently depending on their neighbours?

Despite the number of examples that correlate interspecies interactions in polymicrobial infections with variations in pathogenicity and antibiotic susceptibility of individual organisms, antibiotic therapies are selected to target the most relevant pathogen, with no consideration of the consequences that the presence of other bacterial species may have in the pathogenicity and response to antimicrobial agents. In this issue of Virulence, Garcia-Perez et al. [ 10 ] applied replica plating of used wound dressings to assess the topography of distinct S. aureus types in chronic wounds of patients with the genetic blistering disease epidermolysis bullosa, which is characterized by the development of chronic wounds upon simple mechanical trauma. This approach led to the identification of two strains of S. aureus coexisting with Bacillus thuringiensis and Klebsiella oxytoca. S. aureus is highly prevalent in chronic wound infections, whereas B. thuringiensis and K. oxytoca are regarded as opportunistic pathogens. These bacterial species did not inhibit each other's growth under laboratory conditions, suggesting that they do not compete through the production of inhibitory compounds. Using a top-down proteomic approach to explore the inherent relationships between these co-existing bacteria, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis or K. oxytoca were characterized by Mass Spectrometry.

Antibiofilm activities of norharmane and its derivatives against Escherichia coli O157:H7 and other bacteria.

BACKGROUND: Bacterial biofilms exhibit reduced sensitivity to conventional antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. HYPOTHESIS: The antibiofilm approach using plant alkaloids provides an alternative to antibiotic strategies. STUDY DESIGN: In this study, the antibiofilm activities of various plant alkaloids were investigated against enterohemorrhagic Escherichia coli O157:H7 and Pseudomonas aeruginosa. In the subsequent investigation, the effects of five norharmane derivatives were investigated. RESULT: Harmaline significantly inhibited biofilm formation by E. coli O157:H7, P. aeruginosa PAO1, P. aeruginosa PA14, and Klebsiella oxytoca, and norharmane (ß-carboline) was found to have antibiofilm activity. It was also found that functional groups at the C-1 and C-7 positions of norharmane could play important roles in its antibiofilm activity. Confocal and electron microscopic observations confirmed biofilm inhibition by harmaline and norharmane, and both reduced fimbriae production and swarming and swimming motilities. Furthermore, harmaline and norharmane attenuated the virulence of E. coli O157:H7 in a Caenorhabditis elegans nematode model. CONCLUSION: These findings strongly suggest that harmaline and norharmane could have potential use in antibiofilm strategy against persistent bacterial infections.

Multiple Dictyostelid Species Destroy Biofilms of Klebsiella oxytoca and Other Gram Negative Species.

Dictyostelids are free-living phagocytes that feed on bacteria in diverse habitats. When bacterial prey is in short supply or depleted, they undergo multicellular development culminating in the formation of dormant spores. In this work, we tested isolates representing four dictyostelid species from two genera (Dictyostelium and Polysphondylium) for the potential to feed on biofilms preformed on glass and polycarbonate surfaces. The abilities of dictyostelids were monitored for three hallmarks of activity: 1) spore germination on biofilms, 2) predation on biofilm enmeshed bacteria by phagocytic cells and 3) characteristic stages of multicellular development (streaming and fructification). We found that all dictyostelid isolates tested could feed on biofilm enmeshed bacteria produced by human and plant pathogens: Klebsiella oxytoca, Pseudomonas aeruginosa, Pseudomonas syringae, Erwinia amylovora 1189 (biofilm former) and E. amylovora 1189 Δams (biofilm deficient mutant). However, when dictyostelids were fed planktonic E. amylovora Δams the bacterial cells exhibited an increased susceptibility to predation by one of the two dictyostelid strains they were tested against. Taken together, the qualitative and quantitative data presented here suggest that dictyostelids have preferences in bacterial prey which affects their efficiency of feeding on bacterial biofilms.

Characterization and genome analysis of novel bacteriophages infecting the opportunistic human pathogens Klebsiella oxytoca and K. pneumoniae.

Klebsiella is a genus of well-known opportunistic human pathogens that are associated with diabetes mellitus and chronic pulmonary obstruction; however, this pathogen is often resistant to multiple drugs. To control this pathogen, two Klebsiella-infecting phages, K. oxytoca phage PKO111 and K. pneumoniae phage PKP126, were isolated from a sewage sample. Analysis of their host range revealed that they infect K. pneumoniae and K. oxytoca, suggesting host specificity for members of the genus Klebsiella. Stability tests confirmed that the phages are stable under various temperature (4 to 60 °C) and pH (3 to 11) conditions. A challenge assay showed that PKO111 and PKP126 inhibit growth of their host strains by 2 log and 4 log, respectively. Complete genome sequencing of the phages revealed that their genome sizes are quite different (168,758 bp for PKO111 and 50,934 bp for PKP126). Their genome annotation results showed that they have no human virulence-related genes, an important safety consideration. In addition, no lysogen-formation gene cluster was detected in either phage genome, suggesting that they are both virulent phages in their bacterial hosts. Based on these results, PKO111 and PKP126 may be good candidates for development of biocontrol agents against members of the genus Klebsiella for therapeutic purposes. A comparative analysis of tail-associated gene clusters of PKO111 and PKP126 revealed relatively low homology, suggesting that they might differ in the way they recognize and infect their specific hosts.

Bacteria cell properties and grain size impact on bacteria transport and deposition in porous media.

The simultaneous role of bacteria cell properties and porous media grain size on bacteria transport and deposition behavior was investigated in this study. Transport column experiments and numerical HYDRUS-1D simulations of three bacteria with different cell properties (Escherichia coli, Klebsiella oxytoca, and Rhodococcus rhodochrous) were carried out on two sandy media with different grain sizes, under saturated steady state flow conditions. Each bacterium was characterized by cell size and shape, cell motility, electrophoretic mobility, zeta potential, hydrophobicity and potential of interaction with the sand surface. Cell characteristics affected bacteria transport behavior in the fine sand, but similar bacteria breakthroughs and retardation factors observed in the coarse sand, indicated that bacteria transport was more depended on grain size than on bacteria cell properties. Retention decreased with increasing hydrophobicity and increased with increasing electrophoretic mobility of bacteria for both sand. The increasing sand grain size resulted in a decrease of bacteria retention, except for the motile E. coli, indicating that retention of this strain was more dependent on cell motility than on the sand grain size. Bacteria deposition coefficients obtained from numerical simulations of the retention profiles indicated that straining was an important mechanism affecting bacteria deposition of E. coli and Klebsiella sp., in the fine sand, but the attachment had the same importance as straining for R. rhodochrous. The results obtained in the coarse sand did not permit to discriminate the predominant mechanism of bacteria deposition and the relative implication of bacteria cell properties of this process.

Low-dose irradiation affects the functional behavior of oral microbiota in the context of mucositis.

The role of host-microbe interactions in the pathobiology of oral mucositis is still unclear; therefore, this study aimed to unravel the effect of irradiation on behavioral characteristics of oral microbial species in the context of mucositis. Using various experimental in vitro setups, the effects of irradiation on growth and biofilm formation of two Candida spp., Streptococcus salivarius and Klebsiella oxytoca in different culture conditions were evaluated. Irradiation did not affect growth of planktonic cells, but reduced the number of K. oxytoca cells in newly formed biofilms cultured in static conditions. Biofilm formation of K. oxytoca and Candida glabrata was affected by irradiation and depended on the culturing conditions. In the presence of mucins, these effects were lost, indicating the protective nature of mucins. Furthermore, the Galleria melonella model was used to study effects on microbial virulence. Irradiated K. oxytoca microbes were more virulent in G. melonella larvae compared to the nonirradiated ones. Our data indicate that low-dose irradiation can have an impact on functional characteristics of microbial species. Screening for pathogens like K. oxytoca in the context of mucosits could be useful to allow early detection and immediate intervention.

Bioresorbable ureteral stents from natural origin polymers.

In this work, stents were produced from natural origin polysaccharides. Alginate, gellan gum, and a blend of these with gelatin were used to produce hollow tube (stents) following a combination of templated gelation and critical point carbon dioxide drying. Morphological analysis of the surface of the stents was carried out by scanning electron microscopy. Indwelling time, encrustation, and stability of the stents in artificial urine solution was carried out up to 60 days of immersion. In vitro studies carried out with simulated urine demonstrated that the tubes present a high fluid uptake ability, about 1000%. Despite this, the materials are able to maintain their shape and do not present an extensive swelling behavior. The bioresorption profile was observed to be highly dependent on the composition of the stent and it can be tuned. Complete dissolution of the materials may occur between 14 and 60 days. Additionally, no encrustation was observed within the tested timeframe. The ability to resist bacterial adherence was evaluated with Gram-positive Staphylococcus aureus and two Gram-negatives Escherichia coli DH5 alpha and Klebsiella oxytoca. For K. oxytoca, no differences were observed in comparison with a commercial stent (Biosoft(®) duo, Porges), although, for S. aureus all tested compositions had a higher inhibition of bacterial adhesion compared to the commercial stents. In case of E. coli, the addition of gelatin to the formulations reduced the bacterial adhesion in a highly significant manner compared to the commercial stents. The stents produced by the developed technology fulfill the requirements for ureteral stents and will contribute in the development of biocompatible and bioresorbable urinary stents.

Functional optimization of gene clusters by combinatorial design and assembly.

Large microbial gene clusters encode useful functions, including energy utilization and natural product biosynthesis, but genetic manipulation of such systems is slow, difficult and complicated by complex regulation. We exploit the modularity of a refactored Klebsiella oxytoca nitrogen fixation (nif) gene cluster (16 genes, 103 parts) to build genetic permutations that could not be achieved by starting from the wild-type cluster. Constraint-based combinatorial design and DNA assembly are used to build libraries of radically different cluster architectures by varying part choice, gene order, gene orientation and operon occupancy. We construct 84 variants of the nifUSVWZM operon, 145 variants of the nifHDKY operon, 155 variants of the nifHDKYENJ operon and 122 variants of the complete 16-gene pathway. The performance and behavior of these variants are characterized by nitrogenase assay and strand-specific RNA sequencing (RNA-seq), and the results are incorporated into subsequent design cycles. We have produced a fully synthetic cluster that recovers 57% of wild-type activity. Our approach allows the performance of genetic parts to be quantified simultaneously in hundreds of genetic contexts. This parallelized design-build-test-learn cycle, which can access previously unattainable regions of genetic space, should provide a useful, fast tool for genetic optimization and hypothesis testing.

Metabolic engineering of Klebsiella oxytoca M5a1 to produce optically pure D-lactate in mineral salts medium.

Klebsiella oxytoca strains were constructed to produce optical pure d-lactate by pH-controlled batch fermentation in mineral salts medium. The alcohol dehydrogenase gene, adhE, and the phospho-transacetylase/acetate kinase A genes, pta-ackA, were deleted from the wild type. KMS002 (ΔadhE) and KMS004 (ΔadhE Δpta-ackA) exhibited d-lactate production as a primary pathway for the regeneration of NAD(+). Both strains produced 11-13 g/L of d-lactate in medium containing 2% (w/v) glucose with yields of 0.64-0.71 g/g glucose used. In sugarcane molasses, KMS002 and KMS004 produced 22-24 g/L of d-lactate with yields of 0.80-0.87 g/g total sugars utilized. Both strains also utilized maltodextrin derived from cassava starch and produced d-lactate at a concentration of 33-34 g/L with yields of 0.91-0.92 g/g maltodextrin utilized. These d-lactate yields are higher than those reported for engineered E. coli strains.

Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation.

In Gram-negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston-like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two-hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1-nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self-assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.

[Epidemiological study of Klebsiella spp. uropathogenic strains producing extended-spectrum ß-lactamase in a Tunisian university hospital, 2009]. / Epidémiologie des souches de Klebsiella spp. uropathogènes productrices de ß-lactamases à spectre élargi dans un hôpital universitaire Tunisien, 2009.

OBJECTIVE: An update on the prevalence of antibiotic resistance in extended-spectrum ß-lactamase among urinary strains of Klebsiella spp. isolated from in-come and out-come patients at University Hospital, Mahdia (Tunisia). PATIENTS AND METHODS: A retrospective survey was made over a period of twelve months (year 2009). It focused on 3564 patients with urinary tract infection confirmed by the Laboratory of Microbiology in the University Hospital, Mahdia. RESULTS: Klebsiella spp. was involved in 5.5% (198/3564) of all cases of urinary tract infections identified. Klebsiella pneumoniae accounted for 94.9% of all Klebsiella (5.1% for Klebsiella oxytoca). The frequency of Klebsiella spp. resistance to fluoroquinolones was 19.2% and to third generation cephalosporins was 22.7%. Forty strains of Klebsiella spp. producing extended-spectrum ß-lactamase witch corresponds to 20.2% of all the Klebsiella. The extended-spectrum ß-lactamase strains with resistance to fluoroquinolones were 67.5% (27/40) or 13.6% of all klebsiella (27/198). No strain was resistant to imipenem. The 27 strains multiresistant (ESBL+FQ resistance) are likely to be carriers of plasmids encoding the ESBL and resistance to fluoroquinolones. CONCLUSION: the resistance of Enterobacteriaceae, including Klebsiella spp, to fluoroquinolones has become a concern both in hospital in community medicine. The advent of this resistance mechanism involves a more rational use of fluoroquinolones, especially as first-line treatment of urinary tract infections.

Isolation and characterization of Klebsiella oxytoca strain degrading crude oil from a Tunisian off-shore oil field.

A facultatively anaerobic, Gram-negative, mesophilic, moderately halotolerant, non-motile, and non-sporulated bacterium, designated strain BSC5 was isolated from an off-shore "Sercina" oil field, located near the Kerkennah island, Tunisia. Yeast extract was not required for growth. Phenotypic characteristics and phylogenetic analysis of the 16S rRNA gene sequence of strain BSC5 revealed that it was related to members of the genus Klebsiella, being most closely related to the type strain of K. oxytoca (99% sequence similarity). Strain BSC5 was capable of using aerobically the crude oil as substrate growth. The growth of strain BSC5 on crude oil was followed by measuring the OD(600 nm) and by enumeration of viable cells at different culture's time. GC-MS analysis showed that strain BSC5 was capable of degrading a wide range of aliphatic hydrocarbons from C(13) to C(30) . The biodegradation rate for n -alkanes reached 44% and 75%, after 20 and 45 days of incubation, respectively. Addition of the synthetic surfactant, Tween 80, accelerated the crude oil degradation. The biodegradation rate for n -alkanes reached 61% and 98%, after 20 and 45 days of incubation, respectively. Moreover, three aromatic compounds, p -hydroxybenzoate, protocatechuate and gentisate, were metabolized completely by strain BSC5 after 24 h, under aerobic conditions.

Green fluorescent protein (GFP)-labeling of enterobacteria associated with fruit flies (Diptera: Tephritidae) and persistence in their natural host Rhagoletis completa Cresson.

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 indicate 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, respectively. 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.