Isolation and genomic characterization of a pathogenic Providencia rettgeri strain G0519 in turtle Trachemys scripta.

Providencia rettgeri infection has occurred occasionally in aquaculture, but is rare in turtles. Here, a pathogenic P. rettgeri strain G0519 was isolated from a diseased slider turtle (Trachemys scripta) in China, and qPCR assay was established for the RTX toxin (rtxD) gene. Histopathological examination showed that many inflammatory cells were infiltrated into heart, liver and intestine, as well as the necrosis of liver, kidney and spleen. The genome consisted of one circular chromosome (4.493 Mb) and one plasmid (18.8 kb), and predicted to contain 4170 and 19 protein-coding genes, respectively. Multiple pathogenic and virulence factors (e.g., fimbria, adhesion, invasion, toxin, hemolysin, chemotaxis, secretion system), multidrug-resistant genes (e.g., ampC, per-1, oxa-1, sul1, tetR) and a novel genomic resistance island PRI519 were identified. Comparative genome analysis revealed the closest relationship was with P. rettgeri, and with P. heimbachae closer than with other Providencia spp. To our knowledge, this was first report on genomic characterization of multidrug-resistant pathogenic P. rettgeri in cultured turtles.

Epidemiology and Pathogenesis of Providencia alcalifaciens Infections.

Providencia alcalifaciens is a member of the family Enterobacteriaceae that has been commonly implicated as a causative agent of diarrheal infection in humans and animals. Recent outbreaks of P. alcalifaciens in both developing and developed countries have raised public health concerns. Several studies have suggested that P. alcalifaciens can cause diarrhea by invading the intestinal mucosa, although its pathogenicity has not been well established. Often routine laboratory investigations that seek etiological agents of diarrhea do not actively pursue P. alcalifaciens detection. Therefore, routine laboratory diagnosis should be given more attention for better understanding the epidemiology and pathogenicity of P. alcalifaciens.

Oxidative stress and antioxidant responses in Nile tilapia Oreochromis niloticus experimentally infected by Providencia rettgeri.

Bacterial diseases are one of the major problems in freshwater fish culture and have been linked to significant losses and high mortality rate. In this study, Nile tilapia Oreochromis niloticus was infected by Providencia rettgeri to evaluate the oxidative stress and antioxidant responses in the fish tissues. Juvenile Nile tilapia was divided into two groups, as follow: control (uninfected) and experimentally infected with 100 µL of P. rettgeri suspension containing 2.4 × 107 viable cells/fish, and the liver and kidney tissues were collected on days 7 and 14 post-infection (PI). Liver and kidney ROS and lipid peroxidation levels were high in infected fish on day 14 PI compared to control group, while superoxide dismutase activity was lower in liver (days 7 and 14 PI) and kidney (day 14 PI) compared to their respective control groups. Liver and kidney antioxidant capacity against peroxyl radicals, non-proteic, and proteic thiols levels was lower in infected tilapia on day 14 PI compared to control group. Based on these results, P. rettgeri infection may elicit oxidative damage via increased ROS production, decreased ROS elimination and inhibits enzymatic and non-enzymatic antioxidant defense systems; which may contribute directly to disease pathophysiology of infected animals.

Comparison of twice a day and three times a day meropenem administration in elderly patients in a Japanese community hospital.

Meropenem (MEPM) is a broad-spectrum antibiotic prescribed to patients with moderate or severe pneumonia. It is well recognized that appropriate medicine reduces the burden on not only young patients but elderly ones as well. We enrolled 56 patients aged 75 and over who were diagnosed with moderate or severe pneumonia (body temperature: ≧37.5 °C; white blood cell (WBC) count: ≧10,000/µL; C-reactive protein (CRP): ≧4 mg/dL) on the basis of Clinical Evaluation Methods for New Antimicrobial Agents to Treat Respiratory Infections defined by the Japanese Society of Chemotherapy, at the National Hospital Organization Kanazawa Medical Center from January 1, 2007 to May 31, 2010. Forty-two patients were given MEPM twice a day and 14 were given the same drug three times a day in a Japanese community hospital. After four days, the three times a day group showed significant decreases in body temperature, WBC count, and CRP level, which are commonly used indices for evaluating therapeutic effects. Similarly, the twice a day group showed decreases of those indices, and both treatments had no serious adverse effects. Simulation analysis based on the pharmacokinetics-pharmacodynamics (PK/PD) theory revealed that both treatments effectively inhibited the activities of Pneumococcus, Haemophilus influenzae, Providencia stuartii, and Staphylococcus aureus, which are the major bacteria in the patients. In this retrospective study, simulation analysis based on the PK/PD theory revealed that even the twice a day MEPM administration has sufficient effectiveness against pneumonia. It also may pave the way for the use of personalized medicine in the patients.

Evaluation of Providencia rettgeri pathogenicity against laboratory Mediterranean fruit fly strain (Ceratitis capitata).

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is often referred to as the most severe agricultural pest. Its biological control is mainly through the Sterile Insect Technique (SIT). Colonization, mass-rearing conditions and the irradiation process impact the competitiveness of sterile males and disrupt symbiotic associations by favoring some bacterial species and suppressing others. Levels of Providencia species have been shown to fluctuate considerably in the gut of the medfly laboratory strain Vienna 8 under irradiation, increasing by up to 22%. This study aimed to determine the pathogenicity of Providencia rettgeri isolated from the gut of laboratory Vienna 8 medfly strains by examining the effects of 1) two different treatment doses on egg-hatching and development and 2) two infection methodologies (ingestion and injection) of male and female adults according to their mating status. Treatment of eggs with P. rettgeri (2%) significantly decreased the mean egg to pupae recovery rate. Our data showed significant high mortality in flies with both injection and ingestion after 24 hours without any effect of sex. Microbial counts demonstrated that the bacteria could proliferate and replicate in adult flies. There was a significant sex-dependent effect after infection, with mortality decreasing significantly for males more than females. Providencia rettgeri can be considered as a potential pathogen of C. capitata. Mating protected males and females against infection by P. rettgeri by triggering an immune response leading to double the levels of Cecropin being secreted compared to infected virgin adults, thus reducing the virulence of the bacteria.

Emergence of multidrug-resistant Providencia rettgeri isolates co-producing NDM-1 carbapenemase and PER-1 extended-spectrum ß-lactamase causing a first outbreak in Korea.

BACKGROUND: Nosocomial outbreak due to carbapenem-resistant Enterobacteriaceae has become serious challenge to patient treatment and infection control. We describe an outbreak due to a multidrug-resistant Providencia rettgeri from January 2016 to January 2017 at a University Hospital in Seoul, Korea. METHODS: A total of eight non-duplicate P. rettgeri isolates were discovered from urine samples from eight patients having a urinary catheter and admitted in a surgical intensive care unit. The ß-lactamase genes were identified using polymerase chain reaction and direct sequencing, and strain typing was done with pulsed-field gel electrophoresis (PFGE). RESULTS: All isolates showed high-level resistance to extended-spectrum cephalosporins, aztreonam, meropenem, ertapenem, ciprofloxacin, and amikacin. They harbored the blaNDM-1 carbapenemase and the blaPER-1 type extended-spectrum ß-lactamases genes. PFGE revealed that all isolates from eight patients were closely related strains. CONCLUSIONS: The 13-month outbreak ended following reinforcement of infection control measures, including contact isolation precautions and environmental disinfection. This is the first report of an outbreak of a P. rettgeri clinical isolates co-producing NDM-1 and PER-1 ß-lactamase.

The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion.

Providencia stuartii is the most common Providencia species capable of causing human infections. Currently P. stuartii is involved in high incidence of urinary tract infections in catheterized patients. The ability of bacteria to swarm on semisolid (viscous) surfaces and adhere to and invade host cells determines the specificity of the disease pathogenesis and its therapy. In the present study we demonstrated morphological changes of P. stuartii NK cells during migration on the viscous medium and discussed adhesive and invasive properties utilizing the HeLa-M cell line as a host model. To visualize the interaction of P. stuartii NK bacterial cells with eukaryotic cells in vitro scanning electron and confocal microscopy were performed. We found that bacteria P. stuartii NK are able to adhere to and invade HeLa-M epithelial cells and these properties depend on the age of bacterial culture. Also, to invade the host cells the infectious dose of the bacteria is essential. The microphotographs indicate that after incubation of bacterial P. stuartii NK cells together with epithelial cells the bacterial cells both were adhered onto and invaded into the host cells.

Antibiotic resistance and virulence traits of bacterial pathogens from infected freshwater fish, Labeo rohita.

Bacterial infectious diseases are a main dangerous problem in Aquaculture farming. It causes multiple diseases in fish as well as in human being and it has considerable virulence potential. In this connection, the moot of study focus to discriminate bacterial isolates recovered from naturally diseased Labeo rohita fish and their virulent characteristics. Based on the ß-haemolysis factor, four isolates (KADR11, KADR12, KADR13 and KADR14) were selected for further delineation. These bacterial isolates showed high similarity with Providencia rettgeri, Aeromonas sp., Aeromonas sp. and Aeromonas enteropelogenes respectively, using partial 16S r-RNA gene amplification and biochemical characterizations were also supported. The further study investigates the virulence characteristics of isolates showed separation of outer membrane proteins (OMPs) and lipopolysaccharides (LPS) which appeared between 19_80 kDa and 20_100 kDa in SDS_PAGE analysis respectively. All the four strains were complete resistant (100%) to ß-lactam antibiotics. L. rohita were injected intraperitoneally with 0 (control), 2.0 × 104, 2.0 × 105, 2.0 × 106, 2.0 × 107 and 2.0 × 108 cells/fish of Providencia rettgeri KADR11, Aeromonas sp. KADR12, Aeromonas sp. KADR13 and Aeromonas enteropelogenes KADR14 for the determination of lethal dose 50 (LD50) values, which were 2.4 × 107, 4.1 × 105, 2.7 × 107 and 7.4 × 105 cells/fish respectively. The results indicated that isolated strains were possessed the high pathogenic potential for L. rohita.

First case report of Providencia Rettgeri neonatal sepsis.

BACKGROUND: Providencia are gram negative motile rods and is a member of the Enterobacteriaceae family. It consists of five species, namely Providencia alcalifaciens, Providencia rustigianii, Providencia stuartii, Providencia rettgeri and Providencia heimbachae. These are opportunistic pathogens and leads to infections in immunocompromised host. Providencia rettgeri has been associated with the nosocomial infections of the urinary tract and infections of wounds, burns and blood. Providencia rettgeri is very rare cause of neonatal sepsis and we report first case of neonatal late onset sepsis secondary to it. CASE PRESENTATION: A term male infant presented on day 4 of post-natal life with the complaint of decreased appetite, fast respiration and lethargy. The clinical examination showed features of sepsis and shock with chest radiogram showing pneumonia. The infant was started on invasive ventilation, intravenous fluids, antibiotic and inotropes. The blood culture was suggestive of multi-drug resistant P. rettgeri. The antibiotics were changed according to organism antibiotic susceptibility pattern and infant gradually improved and was discharged successfully. CONCLUSION: Providencia rettgeri is a very rare organism to cause neonatal sepsis. The management involves early diagnosis, treatment with appropriate antibiotics and finding the source of infection.

Genomic Plasticity of Multidrug-Resistant NDM-1 Positive Clinical Isolate of Providencia rettgeri.

We performed a detailed whole-genome sequence analysis of Providencia rettgeri H1736, a multidrug-resistant clinical pathogen isolated in Israel in 2011. The objective was to describe the genomic flexibility of this bacterium that has greatly contributed to its pathogenicity. The genome has a chromosome size of 4,609,352 bp with 40.22% GC content. Five plasmids were predicted, as well as other mobile genetic elements (MGEs) including phages, genomic islands, and integrative and conjugative elements. The resistome consisted of a total of 27 different antibiotic resistance genes including blaNDM-1, mostly located on MGEs. Phenotypically, the bacteria displayed resistance to a total of ten different antimicrobial classes. Various features such as metabolic operons (including a novel carbapenem biosynthesis operon) and virulence genes were also borne on the MGEs, making P. rettgeri H1736 significantly different from other P. rettgeri isolates. A large quantity of the genetic diversity that exists in P. rettgeri H1736 was due to extensive horizontal gene transfer events, leading to an enormous presence of MGEs in its genome. Most of these changes contributed toward the pathogenic evolution of this bacterium.

A Case of Wound Infection with Providencia rettgeri and Coincident Gout in a Patient from Guam.

Providencia rettgeri (P. rettgeri) is a ubiquitous organism that is infrequently associated with human disease. Here we report the isolation of this organism from a polymicrobial wound infection resulting from ruptured tophi on a 54-year-old male patient from Guam. We describe the identification and confirmation of this organism, and propose metabolic synergy as a possible mechanism of pathogenesis. To our knowledge, this is the first published report of a wound infection colonized by P. rettgeri from Guam, and the first report to speculate upon the role of bacterial synergy in P. rettgeri pathogenesis.

The complex contributions of genetics and nutrition to immunity in Drosophila melanogaster.

Both malnutrition and undernutrition can lead to compromised immune defense in a diversity of animals, and "nutritional immunology" has been suggested as a means of understanding immunity and determining strategies for fighting infection. The genetic basis for the effects of diet on immunity, however, has been largely unknown. In the present study, we have conducted genome-wide association mapping in Drosophila melanogaster to identify the genetic basis for individual variation in resistance, and for variation in immunological sensitivity to diet (genotype-by-environment interaction, or GxE). D. melanogaster were reared for several generations on either high-glucose or low-glucose diets and then infected with Providencia rettgeri, a natural bacterial pathogen of D. melanogaster. Systemic pathogen load was measured at the peak of infection intensity, and several indicators of nutritional status were taken from uninfected flies reared on each diet. We find that dietary glucose level significantly alters the quality of immune defense, with elevated dietary glucose resulting in higher pathogen loads. The quality of immune defense is genetically variable within the sampled population, and we find genetic variation for immunological sensitivity to dietary glucose (genotype-by-diet interaction). Immune defense was genetically correlated with indicators of metabolic status in flies reared on the high-glucose diet, and we identified multiple genes that explain variation in immune defense, including several that have not been previously implicated in immune response but which are confirmed to alter pathogen load after RNAi knockdown. Our findings emphasize the importance of dietary composition to immune defense and reveal genes outside the conventional "immune system" that can be important in determining susceptibility to infection. Functional variation in these genes is segregating in a natural population, providing the substrate for evolutionary response to pathogen pressure in the context of nutritional environment.

Reference levels for corticosterone and immune function in farmed saltwater crocodiles (Crocodylus porosus) hatchlings using current Code of Practice guidelines.

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83±SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri, whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32±3% for E. coli and 16±4% for P. rettgeri), the differences had disappeared by approximately 9 months old (58±2% and 68±6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice.

Nosocomial dissemination of plasmids carrying blaTEM-24, blaDHA-1, aac(6')-Ib-cr, and qnrA6 in Providencia spp. strains isolated from a Tunisian hospital.

The aim of this study is to report the emergence of IncA/C conjugative plasmids harboring blaTEM-24, blaDHA-1, qnrA6, and aac(6')-Ib-cr genes among Providencia spp. isolates recovered in 2008 in Tunisia. The double-disk synergy test confirmed the phenotype extended-spectrum ß-lactamase (ESBL) in 2 Providencia stuartii and 5 Providencia rettgeri. These ESBLs were coresistant to cefoxitin, chloramphenicol, ofloxacin, and sulfonamides but remained susceptible to imipenem. Three ß-lactamases TEM-2, TEM-24, and DHA-1 were detected. blaTEM-24, blaDHA-1, aac(6')-Ib-cr, and qnrA6 genes were successfully transferred to Escherichia coli strain HB101, and they were found located on the conjugatifs IncA/C plasmids. Genetic relatedness showed similar and different patterns among P. stuartii and P. rettgeri strains, respectively.

Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity.

Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.

Comparative pathology of bacteria in the genus Providencia to a natural host, Drosophila melanogaster.

Bacteria in the genus Providencia are pathogens of many organisms, including humans and insects. We and colleagues have isolated five different strains belonging to four distinct Providencia species as natural infections of Drosophila melanogaster captured in the wild. We found that these isolates vary considerably in pathology to infected D. melanogaster, differing in the level of mortality they cause, their ability to replicate within the host and the level that the fly's immune response is elicited. One interesting bacterium was Providencia sneebia, which causes nearly complete mortality and reaches large numbers in the fly but does not elicit a comparably strong immune response. Through coinfection experiments, we determined that P. sneebia avoids recognition by the immune system. We tested for biofilm formation and replication within D. melanogaster cells as possible mechanisms for P. sneebia escape from host immunity, but did not find evidence for either. D. melanogaster and Providencia provide a powerful system for studying general host-pathogen interactions, and for understanding how the well-studied immune model host D. melanogaster interacts with its natural bacterial pathogens.

Enterocyte-like Caco-2 cells as a model for in vitro studies of diarrhoeagenic Providencia alcalifaciens invasion.

The entry of Providencia alcalifaciens into the enterocyte-like cell line Caco-2 compared to HEp-2 was studied. Of the 22 P. alcalifaciens strains, 13 and 21 were invasive for Caco-2 and HEp-2 cells, respectively. In contrast to HEp-2 cells, P. alcalifaciens was internalised by Caco-2 cells via receptor-mediated endocytosis. Tyrosine kinases play an important role in P. alcalifaciens uptake, also microfilaments and microtubules are engaged in this process. Inhibition of endosome acidification by ammonium chloride did not seem to have any significant effect on P. alcalifaciens invasion. Similarly to Shigella flexnerii, the invasion of Caco-2 cells by these bacteria occurred more effectively through the basolateral pole than through the apical surface of these cells. Plasmid DNA analysis showed the presence of plasmids of 5-172 kb in 13 strains regardless of their invasive ability. The presence of extracellular bacterial protein, most likely a kind of an invasin, is required for the invasion of Caco-2 and HEp-2 cells.

Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster.

Post-mating reduction in immune defence is common in female insects, and a trade-off between mating and immunity could affect the evolution of immunity. In this work, we tested the capacity of virgin and mated female Drosophila melanogaster to defend against infection by four bacterial pathogens. We found that female D. melanogaster suffer post-mating immunosuppression in a pathogen-dependent manner. The effect of mating was seen after infection with two bacterial pathogens (Providencia rettgeri and Providencia alcalifaciens), though not after infection with two other bacteria (Enterococcus faecalis and Pseudomonas aeruginosa). We then asked whether the evolution of post-mating immunosuppression is primarily a 'female' or 'male' trait by assaying for genetic variation among females for the degree of post-mating immune suppression they experience and among males for the level of post-mating immunosuppression they elicit in their mates. We also assayed for an interaction between male and female genotypes to test the specific hypothesis that the evolution of a trade-off between mating and immune defence in females might be being driven by sexual conflict. We found that females, but not males, harbour significant genetic variation for post-mating immunosuppression, and we did not detect an interaction between female and male genotypes. We thus conclude that post-mating immune depression is predominantly a 'female' trait, and find no evidence that it is evolving under sexual conflict.

Identification of a modular pathogenicity island that is widespread among urease-producing uropathogens and shares features with a diverse group of mobile elements.

Pathogenicity islands (PAIs) are a specific group of genomic islands that contribute to genomic variability and virulence of bacterial pathogens. Using a strain-specific comparative genomic hybridization array, we report the identification of a 94-kb PAI, designated ICEPm1, that is common to Proteus mirabilis, Providencia stuartii, and Morganella morganii. These organisms are highly prevalent etiologic agents of catheter-associated urinary tract infections (caUTI), the most common hospital acquired infection. ICEPm1 carries virulence factors that are important for colonization of the urinary tract, including a known toxin (Proteus toxic agglutinin) and the high pathogenicity island of Yersinia spp. In addition, this PAI shares homology and gene organization similar to the PAIs of other bacterial pathogens, several of which have been classified as mobile integrative and conjugative elements (ICEs). Isolates from this study were cultured from patients with caUTI and show identical sequence similarity at three loci within ICEPm1, suggesting its transfer between bacterial genera. Screening for the presence of ICEPm1 among P. mirabilis colonizing isolates showed that ICEPm1 is more prevalent in urine isolates compared to P. mirabilis strains isolated from other body sites (P<0.0001), further suggesting that it contributes to niche specificity and is positively selected for in the urinary tract.