Discovery of a novel symbiotic lineage associated with a hematophagous leech from the genus Haementeria.

Similarly to other strict blood feeders, leeches from the Haementeria genus (Hirudinida: Glossiphoniidae) have established a symbiotic association with bacteria harbored intracellularly in esophageal bacteriomes. Previous genome sequence analyses of these endosymbionts revealed co-divergence with their hosts, a strong genome reduction, and a simplified metabolism largely dedicated to the production of B vitamins, which are nutrients lacking from a blood diet. 'Candidatus Providencia siddallii' has been identified as the obligate nutritional endosymbiont of a monophyletic clade of Mexican and South American Haementeria spp. However, the Haementeria genus includes a sister clade of congeners from Central and South America, where the presence or absence of the aforementioned symbiont taxon remains unknown. In this work, we report on a novel bacterial endosymbiont found in a representative from this Haementeria clade. We found that this symbiont lineage has evolved from within the Pluralibacter genus, known mainly from clinical but also environmental strains. Similarly to Ca. Providencia siddallii, the Haementeria-associated Pluralibacter symbiont displays clear signs of genome reduction, accompanied by an A+T-biased sequence composition. Genomic analysis of its metabolic potential revealed a retention of pathways related to B vitamin biosynthesis, supporting its role as a nutritional endosymbiont. Finally, comparative genomics of both Haementeria symbiont lineages suggests that an ancient Providencia symbiont was likely replaced by the novel Pluralibacter one, thus constituting the first reported case of nutritional symbiont replacement in a leech without morphological changes in the bacteriome. IMPORTANCE: Obligate symbiotic associations with a nutritional base have likely evolved more than once in strict blood-feeding leeches. Unlike those symbioses found in hematophagous arthropods, the nature, identity, and evolutionary history of these remains poorly studied. In this work, we further explored obligate nutritional associations between Haementeria leeches and their microbial symbionts, which led to the unexpected discovery of a novel symbiosis with a member of the Pluralibacter genus. When compared to Providencia siddallii, an obligate nutritional symbiont of other Haementeria leeches, this novel bacterial symbiont shows convergent retention of the metabolic pathways involved in B vitamin biosynthesis. Moreover, the genomic characteristics of this Pluralibacter symbiont suggest a more recent association than that of Pr. siddallii and Haementeria. We conclude that the once-thought stable associations between blood-feeding Glossiphoniidae and their symbionts (i.e., one bacteriome structure, one symbiont lineage) can break down, mirroring symbiont turnover observed in various arthropod lineages.

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.

Genome-based insights into the resistomes and mobilomes of two Providencia rettgeri strains isolated from wound infections in Madagascar.

OBJECTIVES: A molecular analysis was performed of two Providencia rettgeri (P. rettgeri) strains (Pr 297 and Pr 269) collected in 2007 and 2009 from wound swabs of patients admitted to the intensive care units at Joseph Ravoangy Andrianavalona hospital and the Military Hospital in Antananarivo, Madagascar. METHODS: The two P. rettgeri isolates were subjected to susceptibility testing. Whole genome sequencing was performed to characterise the antibiotic resistance genes, genomic islands and mobilomes (integrons, plasmids and insertion sequences). RESULTS: All isolates were found to be multidrug-resistant. Antibiotic-resistant genes described were amongst eight different classes of antimicrobial agents. Thirty insertion sequences and twelve genomic islands were predicted in each genome. Class 1 and class 2 integrons were found in both genomes, with gene cassette regions encompassing arr-2 - cmlA5 - blaOXA-10 - ant (3")-Ia and dfrA1 - sat2 - ant (3")-Ia - orfX, respectively. IncA/C2, ColM and ColE1-like plasmids were described harbouring blaCMY-30, qnrD and aac(6')-Ib-cr4 genes, respectively. Phylogenetic analysis showed that Pr 297 and Pr 269 isolates were genetically identical and clustered with P. rettgeri strains described in the USA and Spain. CONCLUSIONS: It is believed that this is the first molecular characterisation of wound infection pathogens from Madagascan patients and the first description of P. rettgeri co-producing CMY-30, OXA-10 and AAC(6')-Ib-cr4 enzymes. The diversity of the resistance determinants and mobile genetic elements was probably due to extensive horizontal gene transfer events, highlighting the need to conduct further molecular monitoring studies to understand the genomic plasticity of resistant bacteria in Madagascan hospitals.

Providencia entomophila sp. nov., a new bacterial species associated with major olive pests in Tunisia.

Bioprospection for potential microbial biocontrol agents associated with three major insect pests of economic relevance for olive cultivation in the Mediterranean area, namely the olive fly, Bactrocera oleae, the olive moth, Prays oleae, and the olive psyllid, Euphyllura olivina, led to the isolation of several strains of readily cultivable Gram-negative, rod-shaped bacteria from Tunisian olive orchards. Determination of 16S ribosomal RNA encoding sequences identified the bacteria as members of the taxonomic genus Providencia (Enterobacterales; Morganellaceae). A more detailed molecular taxonomic analysis based on a previously established set of protein-encoding marker genes together with DNA-DNA hybridization and metabolic profiling studies led to the conclusion that the new isolates should be organized in a new species within this genus. With reference to their original insect association, the designation "Providencia entomophila" is proposed here for this hypothetical new taxon.

Providencia huaxiensis sp. nov., recovered from a human rectal swab.

Strain WCHPr000369T was recovered from a human rectal swab in China in 2015. Phylogenetic analysis based on its 16S rRNA gene suggested that the strain belonged to the genus Providencia. The genome sequence of the strain had a 77.30-90.43% average nucleotide identity (ANI) and 20.9-41.5 % in silico DNA-DNA hybridization (isDDH) score with those of type strains of known Providencia species. The ANI and isDDH values indicated that the strain was likely to belong to a new species. Multi-locus sequence analysis on the fusA, lepA, leuS, gyrB and ileS housekeeping genes also revealed that the strain was distinct from any previously described species of the genus Providencia. Strain WCHPr000369T could be distinguished from all known Providencia species by the combination of positive urease reaction and the ability to utilize citrate. Genotypic and phenotypic characteristics from this study indicated that strain WCHPr000369T should be considered to represent a novel species of the genus Providencia, for which the name Providencia huaxiensis sp. nov. is proposed. The type strain is WCHPr000369T (=GDMCC1.1382T=KCTC 62577T).

Dissemination of NDM-1 carbapenemase-producer Providencia stuartii strains in Romanian hospitals: a multicentre study.

Several Romanian hospitals have noted increasing isolation of Providencia stuartii strains in recent years, with an alarming rate of carbapenem resistance. In order to provide molecular epidemiological data regarding their dissemination, 77 P. stuartii strains collected from five hospitals located in different regions of Romania were analysed. All strains harboured IncA/C plasmid, and 67 carried the blaNDM-1 gene. Six clonal clusters were differentiated by pulsed-field gel electrophoresis. The predominant subtype was found in all five hospitals. Our study highlights the need for efficient infection-control measures, the optimization of antibiotic use and the targeted surveillance for carbapenemase-producing P. stuartii.

Development of a molecular serotyping scheme and a multiplexed luminex-based array for Providencia.

Providencia is an opportunistic human pathogen that belongs to the Enterobacteriaceae family. The bacterial cell surface O-antigen is one of the most structurally variable cell constituents and serves as a basis for serotyping gram-negative bacteria. In this work, the genomes of 12 Providencia strains were sequenced, and genes driving O-antigen biosynthesis were analyzed. The O-antigen-synthesizing genes of Providencia are located in the O-antigen gene cluster (OGC) between the cpxA and yibK genes. The gene functions predicted in silico agreed with the known O-antigen structures. All clusters were found to contain both wzx and wzy and exhibit a high degree of heterogeneity. Based on the sero-specific genes, we developed a molecular serotyping system to detect 23 serotypes (from the present and previous studies) for the first time. Five Proteus strains, five Morganella strains, five uropathogenic Escherichia coli (UPEC) strains and 32 Providencia strains with other serotypes were used to assess the specificity of our multiplexed Luminex-based array. Five serogroups (O3, O8, O19, O38 and O52 strains) were used to determine the sensitivity of the suspension array. The detection sensitivity was 0.1 ng genomic DNA, 103 CFU/ml in pure culture, or 104 CFU/ml in mock urine specimens. Furthermore, 29 publicly available Providencia genomes (which have not been serotyped) were analyzed, and 23 novel putative OGC types were identified. In total, we identified 35 new OGCs and developed a molecular serotyping system based on the sero-specific genes. The established classification system can support promising applications in basic research, clinical diagnosis, and epidemiological surveillance.

Circulation of a multiresistant, conjugative, IncA/C plasmid within the nosocomial Providencia stuartii population in the Athens area.

The objective of the study is to report a multidrug-resistant outbreak of Providencia stuartii that occurred in inpatients in the Athens area in 2012 resulting from a very successful transmissible A/C multidrug-resistant plasmid. Thirteen multidrug-resistant P. stuartii clinical isolates from 5 hospitals were studied. Molecular typing was performed by pulsed-field gel electrophoresis. Antibiotic resistance genes and their genetic surround were detected by PCR and sequencing. Plasmid analysis included conjugation experiments using liquid cultures, sizing by S1 digestion, and incompatibility replicon typing by PCR. Isolates were grouped into 2 distinct clonal types A and B, exhibiting similarity less than 70%. Isolates of type A were recovered from patients hospitalized in 4 different hospitals with no obvious epidemiological linkage, while isolates of type B were recovered from patients treated in a single hospital. Both clonal types harbored a conjugative plasmid of 130 bp and IncA/C replicon type carrying 5 ß-lactamase genes bla(SHV-5), bla(VEB-1), bla(VIM-1), bla(OXA-10), and bla(TEM-1) and aminoglycosides resistant determinants. All ß-lactamase genes were included in stable structures as IS26, IS1999, and In-e541. The current plasmid seemed to have many common determinants with previously reported plasmids derived from P. stuartii and Proteus mirabilis clinical isolates and exhibited the ability to circulate in nosocomial bacterial populations.

Emergence of a pandrug-resistant VIM-1-producing Providencia stuartii clonal strain causing an outbreak in a Greek intensive care unit.

Here we describe an outbreak caused by a pandrug-resistant Providencia stuartii strain involving 15 critically ill patients in a Greek intensive care unit (ICU) during September-November 2011. All isolates harboured the blaVIM-1 gene and a class 1 integron structure of 1913 bp as well as blaSHV-5 and blaTEM-1. Pulsed-field gel electrophoresis (PFGE) demonstrated that isolates from all 15 patients belonged to a single P. stuartii clonal type. As all of the infected patients were hospitalised during overlapping time periods, horizontal intra-ICU transmission was considered as the main route for the dissemination of the outbreak strain. The outbreak ended following reinforcement of infection control measures, including implementation of additional barrier precautions for infected patients.

First characterization of a Providencia stuartii clinical isolate from a Tunisian intensive care unit coproducing VEB-1-a, OXA-2, qnrA6 and aac(6')-Ib-cr determinants.

A clinical Providencia stuartii isolate SM662 was recovered from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. This isolate was resistant to penicillins, cephalosporins, aminoglycosides and fluoroquinolones. A marked in vitro synergy between ceftazidime or cefotaxime and amoxicillin-clavulanic acid on Mueller-Hinton agar plates suggested the presence of an extended-spectrum-ß-lactamase. In addition, an unusual synergy was found between cefepime or aztreonam, and cefoxitin or imipenem on a double disk synergy test suggesting a VEB-type extended-spectrum-ß-lactamase. The characterization of ß-lactamases and associated resistance genes was performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Two ß-lactamases bands with pI values of 5.4 and 7.7, which were matched to TEM-1, VEB-1-a and OXA-2-like ß-lactamases were detected. The blaVEB-1-a gene was found to be associated with complex genetic structures, including Re elements. These ß-lactamases were not transferred by electroporation or conjugation experiments to the transconjugants and electroporants. Hybridization methods showed that the extended-spectrum-ß-lactamase encoding gene may have a chromosomal localization. The isolate SM662 produced the quinolone resistance determinants qnrA6 and aac(6')-Ib-cr which were successfully transferred. The detection of an associated chromosomal quinolone resistance revealed the presence of a gyrA mutation at codon 83 (Ser83Ile). This is the first report of the linkage VEB-1-a/OXA-2-like in P. stuartii associated with the description of qnrA6 and aac(6')-Ib-cr genes in this isolate.

Pathogenic Providencia alcalifaciens strain that causes fatal hemorrhagic pneumonia in piglets.

Investigation of a serious pig disease with high mortality and typical lung lesions yielded a bacterial isolate identified as Providencia alcalifaciens based on the 16S ribosomal DNA sequence analysis. The pathogenicity of this bacterial isolate was confirmed in piglets and mice. The bacterial strain caused the typical illness in piglets, which suffered serious dyspnea and hemorrhagic pneumonia. The drug resistance spectrum of the bacterium was also determined. The results indicated that the isolate is resistant to 12 antibiotics and intermediately resistant to 10 antibiotics out of the 34 antibiotics tested. The current study is the first to report a serious lung disease in piglets caused by a multidrug resistant P. alcalifaciens isolate, which should be given more attention during surveillance and diagnostics.

Characterization of a new Providencia sp. strain X1 producing multiple xylanases on wheat bran.

Providencia sp. strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposing site. Strain X1 produced cellulase-free extracellular xylanase, which was higher in wheat bran medium than in xylan medium, when cultivated at pH 8.0 and 35°C. Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran and birchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33; 35; 40; 48; 60; 75; and 95 kDa and 33 and 44 kDa, respectively. The crude xylanases were produced on wheat bran medium and showed optimum activity at pH 9.0 and 60°C. The thermotolerance studies showed activity retention of 100% and 85% at 40°C and 60°C after 30 min preincubation at pH 9.0. It was tolerant to lignin, ferulic acid, syringic acid, and guaiacol and retained 90% activity after ethanol treatment. The enzyme preparation was also tolerant to methanol and acetone and showed good activity retention in the presence of metal ions such as Fe2+, Mg2+, Zn2+, and Ca2+. The crude enzyme preparation was classified as endoxylanase based on the product pattern of xylan hydrolysis. Pretreatment of kraft pulp with crude xylanases for 3 h at 60°C led to a decrease in kappa number by 28.5%. The properties of present xylanases make them potentially useful for industrial applications.

Providencia thailandensis sp. nov., isolated from seafood processing wastewater.

The bacterial strain C1112(T) was isolated from seafood processing wastewater collected from a treatment pond of the seafood factory in Songkhla Province, Thailand. Phylogenetic analysis based on concatenated sequences from the 16S rRNA gene and five housekeeping genes, fusA, lepA, leuS, gyrB and ileS respectively showed that the strain C1112(T) belonged to the genus Providencia, and share 91.75% similarity with P. stuartii DSM 4539(T). DNA-DNA hybridization between the strain C1112(T) and P. stuartii KCTC 2568(T) was 48.1% relatedness. Moreover, some results from biochemical properties indicated that the strain C1112(T) was distinguished from the phylogenetically closest relatives. The major fatty acids of the strain C1112(T) were C16:0, iso-C15:0, C14:0 and C17:0 cyclo and the DNA G+C content was 41 mol%. Based on the genotypic and phenotypic considerations, it should be classified as a novel species of the genus Providencia for which the name Providencia thailandensis sp. nov. is proposed. The type strain is C1112(T) (= KCTC 23281(T) =NBRC 106720(T)).

Renal abscess caused by a Providencia stuartii isolate biochemically misidentified as Pasteurella.

Providencia stuartii is associated with urinary tract infection (UTI) in catheterized patients. Here we report an abscess containing P. stuartii in a patient with a history of UTI, renal stones, and stent placement. This organism was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing following biochemical identification as Pasteurella.

Nosocomial dissemination of Providencia stuartii isolates carrying bla OXA-48, bla PER-1, bla CMY-4 and qnrA6 in a Tunisian hospital.

OBJECTIVES: To report an outbreak due to Providencia stuartii isolates carrying bla(OXA-48), bla(PER-1), bla(CMY-4) and qnrA6 in a Tunisian hospital in 2011. METHODS: Eight intensive care unit (ICU) patients infected/colonized by extended-spectrum ß-lactamase (ESBL)-producing P. stuartii between March and May 2011 were included. Molecular epidemiology was studied by PFGE. Antibiotic resistance genes were analysed by PCR and sequencing and the plasmid incompatibility group by a PCR-based replicon typing scheme. RESULTS: Eight patients were colonized with ESBL-producing P. stuartii isolates. All these isolates were clonally related and found to carry bla(OXA-48), bla(PER-1), bla(CMY-4), qnrA6 and aac-6'-Ib genes on the same self-conjugative IncA/C plasmid. The same strain was also cultured from environmental samples in the ICU. All these isolates were susceptible to carbapenems. Only one colonized patient developed P. stuartii pleurisy and was effectively treated with imipenem alone. CONCLUSIONS: This is the first report of an outbreak due to P. stuartii isolates carrying bla(OXA-48) in Tunisia. The simultaneous expression of various resistance genes (bla(OXA-48), bla(CMY-4), bla(PER-1), qnrA and aac-6'-Ib) by P. stuartii isolates is alarming.

Prevalence of Providencia strains among children with diarrhea in Japan.

In the present study, we examined the prevalence of Providencia spp. strains among children with diarrhea in Japan. We developed a Providencia genus-specific polymerase chain reaction (PCR) method, and the specificity and sensitivity was evaluated to be 100% with various bacterial strains including 7 genera and 13 species. Five of 345 samples (1.4%) were positive by PCR using a Providencia genus-specific primer targeting the 16S rRNA gene. The single species Providencia rettgeri was isolated from 4 stool samples of children with diarrhea. The prevalence of Providencia spp. in children with diarrhea in Japan is lower than that previously reported for Japanese travelers abroad with diarrhea.

Nosocomial dissemination of extended-spectrum ß-lactamase VEB-1a-producing Providencia stuartii isolates in a Tunisian hospital.

A collection of 20 multidrug-resistant Providencia stuartii isolates recovered from 2005 to 2009 at the Military Hospital of Tunis, Tunisia, was analysed. They all expressed the extended-spectrum ß-lactamase (ESBL) VEB-1a. The bla (VEB-1a) gene was plasmid-located and it was associated with complex genetic structures, including Re elements. Pulsed-field gel electrophoresis (PFGE) revealed a clonal relationship between all of these isolates. This study identified a nosocomial dissemination of an ESBL-producing P. stuartii clone in a Tunisian hospital over a long period of time.

Phylogenetic analysis of the genera Proteus, Morganella and Providencia by comparison of rpoB gene sequences of type and clinical strains suggests the reclassification of Proteus myxofaciens in a new genus, Cosenzaea gen. nov., as Cosenzaea myxofaciens comb. nov.

Phylogenetic analysis of partial rpoB gene sequences of type and clinical strains belonging to different 16S rRNA gene-fingerprinting ribogroups within 11 species of enterobacteria of the genera Proteus, Morganella and Providencia was performed and allowed the definition of rpoB clades, supported by high bootstrap values and confirmed by ≥2.5 % nucleotide divergence. None of the resulting clades included strains belonging to different species and the majority of the species were confirmed as discrete and homogeneous. However, more than one distinct rpoB clade could be defined among strains belonging to the species Proteus vulgaris (two clades), Providencia alcalifaciens (two clades) and Providencia rettgeri (three clades), suggesting that some strains represent novel species according to the genotypes outlined by rpoB gene sequence analysis. Percentage differences between the rpoB gene sequence of the type strain of Proteus myxofaciens and other members of the same genus (17.3-18.9 %) were similar to those calculated amongst strains of the genus Providencia (16.4-18.7 %), suggesting a genetic distance at the genus-level between Proteus myxofaciens and the rest of the Proteus-Providencia group. Proteus myxofaciens therefore represents a member of a new genus, for which the name Cosenzaea gen. nov., is proposed.