O-serotyping Providencia alcalifaciens.

The O-serotyping scheme for Providencia was tested on Providencia alcalifaciens isolates collected mostly from two hospitals. The specificites of the somatic (O) antigens of P. alcalifaciens were found to be different from those of Providencia stuartii, and separation of the Providencia typing scheme to allow separate typing of each species led to more efficient typing. All but 4 of 86 isolates were typable. Eighteen serotypes occurred among 53 typable isolates obtained from a pediatric hospital, and 11 occurred among 19 isolates from a general hospital. Thirty-two percent of the isolates from the pediatric hospital belonged to serotype O3, the most frequently isolated and most widely distributed type. The use of the serotyping scheme for P. alcalifaciens is advocated for further studies to examine strains of the species for enteropathogenic types.     

Molecular characterizations of cytolethal distending toxin produced by Providencia alcalifaciens strains isolated from patients with diarrhea

Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides--CdtA, CdtB, and CdtC--encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206-215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G(2)/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.     

Cloning of urease gene sequences from Providencia stuartii.

Providencia stuartii was the most prevalent isolate recovered from urine specimens taken weekly over a 1-year period from 51 nursing home patients with urinary catheters in place. Thirty percent of the isolates were urease positive. Urease, which is implicated in renal stone formation, was shown to be transmissible on an 82-kilobase conjugative plasmid in one isolate. Plasmid DNA isolated from this strain was digested with EcoRI, ligated into the EcoRI site of pBR322, and used to transform Escherichia coli HB101. Ampicillin-resistant clones were replica plated onto urea segregation agar, and a urease-positive clone, designated pMID101, was isolated. Recombinant and native urease from cell lysates had identical electrophoretic mobilities on nondenaturing polyacrylamide urease activity gels. The native enzyme was induced fourfold when cells were grown in the presence of 0.1% urea and had a km of 9.4 mM and a Vmax of 3.2 mumol of NH3 per min per mg of protein. Its molecular weight was estimated to be 375,000 +/- 35,000 by Sephacryl S-300 chromatography. The enzyme was cytoplasmic in P. stuartii, was inhibited in vitro by hydroxyurea, acetohydroxamic acid, and EDTA, and appears to have a complex subunit structure and a unique molecular size within genera of the Proteeae tribe.     

Providencia rustigianii: a new species in the family Enterobacteriaceae formerly known as Providencia alcalifaciens biogroup 3.

The name Providencia rustigianii sp. nov. is proposed for a group of organisms previously known as Providencia alcalifaciens biogroup 3. By DNA hybridization, strains of P. rustigianii were 81 to 99% related to each other at 60 degrees C, but only 44 to 49% related to P. alcalifaciens biogroups 1 and 2 and 26 to 33% related to Providencia stuartii. P. rustigianii could be differentiated from P. alcalifaciens and P. stuartii by simple biochemical tests. P. rustigianii produced acid from D-galactose but not from trehalose; P. stuartii produced acid from both; and P. alcalifaciens produced acid from neither. P. rustigianii could be distinguished from Providencia rettgeri (formerly Proteus rettgeri) by urea hydrolysis and acid production from D-arabitol; P. rustigianii was negative for these two tests, but P. rettgeri was positive. Strains of P. rustiganii were 32 to 34% related to strains of P. rettgeri. Three of the 11 strains of P. rustigianii were isolated from stools, but the sources of the other isolates are unknown. Three strains (27%) were sensitive to colistin, and 82 to 100% were sensitive to ampicillin, carbenicillin, cephalothin, gentamicin, kanamycin, nalidixic acid, streptomycin, and tetracycline. Strain ATCC 33673 (CDC no. 0132-68) is the type strain for this species.     

Sensitivity of urine-grown cells of Providencia stuartii to antiseptics.

Urine-grown cultures of 23 clinical isolates of Gram-negative bacteria having a range of minimum inhibitory concentration values for chorhexidine were challenged with various concentrations of this antiseptic. The results suggest that cells of Providencia stuartii, in particular, exhibit a considerable degree of resistance to chlorhexidine under these conditions, concentrations of up to 10 000--20 000 microgram/ml of urine being necessary to produce complete loss of viability of such cultures. Of the other two antiseptics tested, phenoxyethanol proved to be the more effective, the recommended use concentration of 2% v/v producing reductions in viable counts of greater than six logarithms in all the strains examined. It is suggested that phenoxyethanol may be a suitable alternative to the cationic agents for use in antiseptic policies for bladder management of urinary tract infections with Providencia stuartii.     

Variable phenotypes of Providencia stuartii due to plasmid-encoded traits.

Weekly urine specimens from 51 long-term catheterized patients yielded 699 isolates of Providencia stuartii. Urease-positive strains represented 23.7% (166) of the isolates, sucrose-positive strains represented 24.5% (171), and lactose-utilizing strains represented 0.7% (5). Urease and sucrose traits were transferred by conjugation to Escherichia coli via an 82-kilobase plasmid; lactose fermentation was transferred by a 150-kilobase plasmid.     

Antibiotic resistance in Providencia stuartii isolated in hospitals.

A total of 238 isolates of Providencia stuartii obtained from infected patients in six Dublin hospitals were grouped by using serological and bacteriocin typing methods and tested for sensitivity to a number of antimicrobial agents. Most isolates were resistant to several of these agents. Resistance to tetracycline, resistance to penicillin, resistance to polymyxin, and probably resistance to nitrofurantoin was intrinsic. Plasmid screening coupled with resistance transfer studies showed that both chromosome-encoded and plasmid-coded resistance mechanisms were clinically important. Ampicillin resistance was both chromosomally and plasmid encoded, whereas resistance to kanamycin and resistance to carbenicillin were exclusively plasmid encoded. Gentamicin resistance was more common than kanamycin resistance, and although gentamicin-resistant strains contained aminoglycoside acetyltransferase activity, no association could be demonstrated with plasmid deoxyribonucleic acid in the strains tested. Unlike minimal inhibitory concentrations for kanamycin, minimal inhibitory concentrations for gentamicin varied over a wide range. P. stuartii isolated obtained from several different countries were tested for comparison. As a group, these strains were less resistant, but they did exhibit similar resistance properties.     

Commercial identification systems often fail to identify Providencia stuartii.

We tested 145 clinical isolates in an attempt to evaluate some of the most widely used commercial identification systems in Europe in terms of their ability to identify Providencia strains. Two manual miniaturized systems (API 20E and Enterotube II) and three mechanized-automated systems (Cobas-Bact, Sceptor System, and Titertek-Enterobac-Rapid Automated System) were evaluated. Providencia alcalifaciens and Providencia rettgeri strains were correctly identified by all systems in all cases, and in most cases identification was achieved without the aid of supplementary tube tests. By contrast, Providencia stuartii was identified without the aid of supplementary tube tests for only 42.5% (API 20E), 37.5% (Enterotube), 68.7% (Sceptor), and 71.2% (Cobas-Bact) of the isolates. The overall misidentification rates were 16.3, 11.3, 11.3, and 10%, respectively. The Titertek-Enterobac-Rapid Automated System failed to identify only 1 of 80 strains (1.3%) and required supplementary tests in 2 other cases (2.5%). Since four of the multitest systems examined often failed to correctly identify P. stuartii, we conclude that supplementary conventional tube tests should always be used to distinguish this species from the other taxa of the Proteeae tribe.     

Meningitis due to Providencia stuartii

In this report, we present a case of postneurosurgical meningitis due to Providencia stuartii, which was treated successfully with meropenem therapy lasting 21 days.     

O serotyping of Providencia stuartii isolates collected from twelve hospitals.

A collection of 829 isolates of Providencia stuartii, mostly from urological specimens of patients in 12 hospitals, were O serotyped. Hospitals varied in serotype distribution, but most isolates (97%) fell into one or another of 14 O types of P. stuartii. One type (O63) was found in 10 hospitals, and six types (O4, O17, O25, O52, O55, O56) were found in 5 or more hospitals. These seven types were more common than others and included 753 (91%) of the isolates. Only four isolates agglutinated in Providencia alcalifaciens antisera and, for increased efficiency in serotyping, it is recommended that separate schemes be employed for P. stuartii and P. alcalifaciens. Strains endemic in different hospitals may differ in serotype and give rise to nosocomial infections that are clinically recognizable when infections occur in obvious clusters. Nosocomial infections occurring in low frequency among patients not located close to each other in the hospital may be detected with the aid of serotyping.     

Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4 degrees C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4 degrees C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 degrees C dissociated from MVM when the temperature was raised to 37 degrees C, but reassociated when the temperature was again brought to 4 degrees C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.     

Spurious hydrogen sulfide production by Providencia and Escherichia coli species.

Hydrogen sulfide production was noted in two Escherichia coli strands and one Provaidenica alcalifaciens (Proteus inconstans A) strain isolated from clinical stool specimens durin the summer of 1979. An investigation into this phenomenon revealed the predence of Eubacterium lentum, an anaerobe, growing in synergism with the Enterobacteriaceae and producing H2s. The implications of this association are discssed with reference to clinical microbiology laboratory practice.     

Isolation of Providencia heimbachae from Human Feces

Providencia heimbachae was first described in 1986. It has been isolated from penguin feces and an aborted bovine fetus. To date, there has been no reported isolation of this organism from human specimens. We now report the isolation of P. heimbachae from the stool of a 23-year-old woman with idiopathic diarrhea. The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of P. heimbachae.     

Molecular analysis of clinical isolates of Providencia alcalifaciens

In an attempt to elucidate the virulence factors and the pathogenic mechanisms of Providencia alcalifaciens, 36 isolates identified in 1994-1995 in Recife city, Brazil were analysed by PCR to investigate the presence of DNA sequences homologous to virulence genes described in other invasive enterobacteria, as well as their ability to invade HeLa cells, their plasmid profiles and antibiotic resistance patterns. The genetic diversity of the isolates was also analysed by RAPD-PCR. No homologous sequences of virulence genes were observed with any of the P. alcalifaciens isolates studied. Ten isolates had no plasmid and 26 harboured one-to-five plasmids of 147-<6.9 kb. Invasion of HeLa cells was observed in only 10 isolates. No correlation between the plasmid content of the strains, their invasion of HeLa cells or their resistance to antimicrobial drugs could be established. The isolates could be distributed into 10 genotypic groups by RAPD-PCR. Considering the genotypic profile and ability to invade HeLa cells, 7 of the 10 invasive isolates belonged to the same genotypic group. The presence of invasive isolates in the same or a related genotypic group suggests the existence of a clonal lineage responsible for the invasiveness.     

Modified MacConkey medium which allows simple and reliable identification of Providencia stuartii.

This work describes a modified MacConkey medium (MCP medium) enabling the simple identification of Providencia stuartii, an emerging nosocomial pathogen. A total of 813 strains, belonging to the families Enterobacteriaceae and Pseudomonadaceae, were tested on MCP medium; all P. stuartii strains were phosphatase positive, as were 97.5% of Morganella morganii strains, in contrast with all other tested organisms. A simple discriminating test, such as the ornithine or citrate test, allowed identification of strains of these species. We have also compared the reliabilities of P. stuartii identification by commercial kits (API 20E system) by using a standard MacConkey or MCP medium. Sixteen and three-tenths percent of P. stuartii strains were misidentified by using the former procedure, while with the latter all strains were correctly identified. Finally, the MCP medium was used over a 6-month period in our routine clinical laboratory. Of a total of 1,278 seeded urine samples from elderly patients, we isolated 103 P. stuartii strains which were all correctly identified by coupling MCP medium and the API 20E system. Seventeen and one-half percent of these strains were misidentified when the API 20E system was used in combination with standard MacConkey medium.