Characterization of first hemin-requiring Pseudomonas aeruginosa small-colony variants from the blood of an octogenarian male-patient with double pneumonitis.

A hemin-requiring Pseudomonas aeruginosa small-colony variant (SCV) was isolated from the blood of an octogenarian male-patient with double pneumonitis. The isolate was capable of growing on both sheep blood and chocolate agars but not on MacConkey agars without blood ingredient. Furthermore, the isolate revealed to grow only around the X-factor impregnated discs when examined using the X and V disc strips. However, not only RapID-NH system but also the VITEK2 system failed to identify the isolate. The isolate was finally identified as P. aeruginosa by the sequence of the 16S rRNA genes and the MALDI-TOF MS analysis. Interestingly, the isolate represented positive reaction for δ-aminolaevulinic acid (ALA)-test despite the requirement of hemin. Detailed analysis indicated that the isolate produced protoporphyrin IX from ALA. Therefore, the reason for the hemin dependence was deduced the dysfunction of hemH-encoded ferrochelatase behaving at the end of biosynthetic pathway of heme. However, the genetic analysis of hemH gene demonstrated no variations of both the DNA and the amino-acid sequences. To the best of our knowledge, this is the first clinical isolation of a hemin-dependent P. aeruginosa SCV from blood.

Notable alkaline tolerance of Kocuria marina isolate from blood of a pediatric patient with continuous intravenous epoprostenol therapy.

This study was the first to describe the hitherto deficiently evaluated alkaline tolerance of Kocuria marina isolate from a pediatric patient with continuous intravenous epoprostenol dosing therapy. Our isolate from blood of a 7-year-old Japanese boy was finally identified as K. marina by the morphological, cultural, and biochemical properties together with the comparative sequence analyses of the 16S rRNA genes. The K. marina isolate, the causative agent of catheter-related blood-stream infection, was not only revealed to be salt tolerant (NaCl 15%), but also demonstrated to be stably survived with no apparent decrease of cell counts for long periods (120 h) in an alkaline environment (pH 8, 9, 10, and 11) at 35 °C. Its remarkable tolerance to the stresses of high alkalinity compared with a clinical Staphylococcus aureus strain should provide consistent interpretation that the environment of high alkalinity (pH 10.2-10.8) measures should be insufficient to inactivate almost all the causative agents including K. marina strains in the solution of epoprostenol (pH 10.4) (Flolan(®), GlaxoSmithKline, Ltd., Tokyo, Japan.). To the best of our knowledge, the first description of the property of being tolerant to high alkalinity that the K. marina isolate exhibited was noteworthy and a useful piece of information. In conclusion, we believe that the present study should be a notification regarding the potential risk of catheter-related blood-stream infections due to K. marina, suggestive of an alkalophile, especially in patients receiving continuous intravenous epoprostenol dosing therapy.

Incidence of Legionella and heterotrophic bacteria in household rainwater tanks in Azumino, Nagano prefecture, Japan.

Many administrative agencies in Japan are encouraging installation of household rainwater-storage tanks for more effective use of natural rainwater. Water samples were collected periodically from 43 rainwater tanks from 40 households and tested for the presence of Legionella species and the extent of heterotrophic bacteria in Azumino city, Nagano prefecture, Japan. PCR assays indicated the presence of Legionella spp. in 12 (30%) of the 43 tank water samples. Attempts were made to identify correlations between PCR positive samples, topography, pH, chemical oxygen demand (COD), atmospheric temperature and the numbers of heterotrophic bacteria. Between June and October, 2012, the numbers of heterotrophic bacteria in rainwater tanks and the values of COD positively correlated with the presence of Legionella species. In most of the Legionella-positive cases, heterotrophic bacterial cell counts were >10(4) CFU/mL. Moreover, Legionella species were less frequently detected when the COD value was >5 mg KMnO(4)/L. Therefore, at least in Azumino, Japan between June and October 2012, both heterotrophic bacterial counts and COD values may be considered index parameters for the presence of Legionella cells in rainwater tanks. Much more accumulation of such data is needed to verify the accuracy of these findings.

Isolation of an X-factor-dependent but porphyrin-positive Escherichia coli from urine of a patient with hemorrhagic cystitis.

An Escherichia coli isolate was recovered from a 92-year-old female patient with urinary tract infection. Gram-stained preparation of the urine sediment manifested some gram-negative rod-shaped cells, and the urine specimen culture yielded nonhemolytic colonies on sheep blood agar plate. However, no visible colonies appeared on modified Drigalski agar plate. The isolate was finally identified as an X-factor-dependent E. coli. The interesting finding was that the isolate revealed a positive reaction for porphyrin test despite the requirement of hemin. This finding suggested that some pyrrol-ring-containing porphyrin compounds or fluorescent porphyrins had been produced as chemical intermediates in the synthetic pathway from δ-amino-levulinic acid (ALA), although the isolate should be devoid of synthesizing hems from ALA. This was the first clinical isolation of such a strain, indicating that the E. coli isolate should possess incomplete synthetic pathways of hems from ALA.

Characterization of CIA-1, an Ambler class A extended-spectrum ß-lactamase from Chryseobacterium indologenes.

An Ambler class A ß-lactamase gene, bla(CIA-1), was cloned from the reference strain Chryseobacterium indologenes ATCC 29897 and expressed in Escherichia coli BL21. The bla(CIA-1) gene encodes a novel extended-spectrum ß-lactamase (ESBL) that shared 68% and 60% identities with the CGA-1 and CME-1 ß-lactamases, respectively. bla(CIA-1)-like genes were detected from clinical isolates. In addition to the metallo-ß-lactamase IND of Ambler class B, C. indologenes has a class A ESBL gene, bla(CIA-1), located on the chromosome.

[Successful isolation of IMP-1 carbapenemase-producing, but not carbapenem-resistant species in Enterobacteriaceae family].

Carbapenemases including Klebsiella pneumoniae carbapenemases (KPC) are widespread among clinical isolates in the family Enterobacteriaceae. In 2008, we isolated 4 IMP-1 metallo-beta-lactamase (MBL) producers of this family having transferable carbapenem-resistance markers. When examined with MicroScan Neg-Combo Panels, all 4 showed imipenem-MIC of either <1 microg/mL or 2 microg/mL, although they were highly resistant to ceftazidime (MIC: >16 microg/mL). When isolates were examined by Sensi-Disc, however, discrepancies were seen in susceptibility testing results against carbapenems, i.e., some strains were susceptible to imipenem but resistant to meropenem. MBL productivity of isolates could be ensured by both sodium mercaptoacetic acid (SMA) and modified Hodge testing. Noted that atypical carbapenemase-producers may be overlooked in routine clinical microbiology laboratory testing, and both SMA disks and modified Hodge tests proved appropriate for accurately detecting such carbapenemase-producers.

[Clinical assessment of novel ChromID ESBL agar plates for detection of ESBL producers in the family Enterobacteriaceae].

Extended-Spectrum beta-Lactamase (ESBL)-producers in the family Enterobacteriaceae are recognized worldwide as nosocomial pathogens, however it is difficult to screen them in the routine laboratory processing. ChromID ESBL agar newly developed for screening ESBL-producing Enterobacteriaceae was released in Japan in April, 2007. We evaluated the clinical assessment of ChromID ESBL agar in routine microbiology laboratory. The 47 strains investigated were clinical isolates belonging to the family Enterobacteriaceae with the MICs of cefpodoxime greater than 2 mug/ml. The 27 ESBL-producers examined were comprising of 19 Escherichia coli, 3 Klebsiella oxytoca, 1 Citrobacter freundii, 3 Enterobacter cloacae, and 1 S. marcescens (ESBL group) and 20 ESBL non-producers consiating of 5 K. oxytoca, 1 Proteus mirabilis, 1 P. vlugaris, 2 Serratia marcescens, 8 C. freundii, 2 Enterobacter cloacae, and 1 E. aerogenes (non-ESBL group). Characterization of beta-lactamase genes was carried out by use of polymerase chain reaction. As the results, the sensitivity and the specificity of ChromID ESBL agar plates after incubation for 18 hours was 100% and 20%, respectively. It should be noted that the values of specificity was extremely low compared with those of the sensitivity. These findings clearly suggested that in cases of utilizing ChromID ESBL agar plates, it should be important to consider its characteristic properties, as even the ESBL-non-producers could grow on these media only when they were resistant to CPDX.

Isolation of a Capnophilic and Extended-Spectrum ß-Lactamase-Producing Proteus mirabilis Strain from the Urine of an Octogenarian Male Patient with Acute Pyelonephritis.

A capnophilic Gram-negative rod-shaped bacterium was recovered from the urine of an octogenarian male patient with acute pyelonephritis. The isolate was found to produce CTX-M-2-type extended-spectrum ß-lactamase. Interestingly, the isolate failed to grow on modified Drigalski (BTB) and MacConkey agar media, even under CO2-enriched atmosphere. Our analysis revealed that the pH-indicator dyes, bromothymol blue, and/or crystal violet that were incorporated into the agar media inhibited the growth of the isolate. Although routine identification methods using Vitek® 2 Compact systems were unsuccessful, the isolate was identified as Proteus mirabilis by 16S rRNA sequencing and MALDI-TOF MS analysis. The carbonic anhydrase (CA) region spanning approximately 2,000 bp upstream to 2,000 bp downstream, which is responsible for the CO2 requirement, was not amplified, which could be attributed to the large-scale deletion or mutation of the DNA sequences containing the CA gene region. In fact, revertants with the ability to grow without CO2 were not detected. However, a revertant that was capable of growing in both BTB and MacConkey agar was detected at frequencies less than 10-9. Therefore, the genes responsible for the highly sensitive reactions of the isolate to pH indicator dyes is not likely to be linked to the CA genes.

In vivo bactericidal activities of Japanese rice-fluid against H. pylori in a Mongolian gerbil model.

PURPOSE: The antibiotic effect of rice-fluid on Helicobacter pylori infection was investigated using a Mongolian gerbil model. METHODS: Gerbils were divided into four groups: H. pylori -infected, rice-fluid-treated animals (group A); H. pylori -infected, untreated animals (group B); uninfected, rice-fluid-treated animals (group C); and uninfected, untreated animals (group D). Group A and B animals were killed 14 weeks after H. pylori infection and group C and D animals were killed at the same age. The stomachs were examined for histology, 5'-bromo-2'-deoxyuridine (BrdU) labeling, and the bacterial burden. Serum anti-H. pylori antibody titers were also tested. RESULTS: The positive incidence of H. pylori -culture was 25 and 84 % in groups A and B, respectively (p<0.01). Both the degree of inflammation and the BrdU labeling index in group A were significantly lower than those in group B. CONCLUSIONS: Rice-fluid showed an antibiotic effect on H. pylori and an anti-inflammatory effect on the H. pylori -associated gastritis.

Third-Generation Cephalosporin-Resistant Non-Typhoidal Salmonella Isolated from Human Feces in Japan.

ß-lactamase genes were detected and characterized from 10 non-typhoidal Salmonella (NTS) clinical isolates resistant to third-generation cephalosporins collected between 2012 and 2014 in Japan. Five strains showed cefotaxime minimum inhibitory concentration (MIC) ≥ 64 µg/ml and positive clavulanic acid inhibition results. The blaCTX-M-2 was detected in 3 strains (serotypes Stanley and Muenchen), whereas blaTEM-52 (serotype Manhattan) and blaSHV-12 (serotype Infantis) were each found in 1 strain. blaCMY-2 was detected in the remaining 5 strains (serotypes Infantis, Rissen, Newport, and Saintpaul) with cefotaxime MICs of 4-32 µg/ml and positive cloxacillin- and 3-aminophenylboronic acid- based inhibition tests. ISEcp1 was located upstream of the blaCMY-2 in 4 strains and of the blaCTX-M-2 in 1 strain. Incompatibility (Inc)A/C, IncP, and IncI1 plasmids were present in the strains harboring blaCMY-2, which were detected predominantly in this study. Acquisition of resistance to third-generation cephalosporins by invasive NTS may limit therapeutic options for severe systemic infections and causing serious public health problems. Though such resistant clinical isolates are still rare in Salmonella species in Japan, our findings reveal the presence of cephem-resistant NTS in food handlers, thus emphasizing the necessity of more systematic nationwide investigations.

First isolation of Dysgonomonas mossii from intestinal juice of a patient with pancreatic cancer.

BACKGROUND: Dysgonomonas species were first designated in 2000. However, clinical infections due to this microorganism have rarely been described. Our aim was to present the first isolation of Dysgonomonas mossii from intestinal juice of a patient with pancreatic cancer. METHODS: Predominantly appearing grayish-white colonies grown on chocolate and sheep blood agar plates were characterized morphologically by Gram stain, biochemically by automated instrument using Vitek II ID-GNB card together with commercially available kit systems, ID-Test HN-20 and API rapid ID 32A32A, and genetically by sequencing the 16S rRNA gene of the organism using a Taq DyeDeoxy Terminator Cycle Sequencing and a model 3100 DNA sequencer instrument. The isolate was further characterized by antimicrobial susceptibility using MicroFast 4J Panels and additional biochemical and physiological properties. RESULTS: The isolate was finally identified as D. mossii from the findings of the morphological, cultural, and biochemical properties together with the comparative sequence of the 16S rRNA genes. The isolate was highly susceptible to many antibiotics but resistant to penicillins and cephems. CONCLUSIONS: As D. mossii was rarely encountered in the clinical microbiology laboratory, it may be misidentified as an X-factor-dependent Haemophilus species due to its negative result for the porphyrin test. Accumulation of the case reports with the isolation of this species is expected to elucidate the infections due to D. mossii. The presence of D. mossii caused no significant clinical infection despite repeated isolations, as the patient had no conspicuous abdominal complaints. However, our report is a noteworthy and useful piece of information.

In vitro bactericidal activities of Japanese rice-fluid against Helicobacter pylori strains.

BACKGROUND: Helicobacter pylori has now been widely recognized as a causative agent of gastroduodenal diseases. The development of safer anti- H. pylori compounds is desirable due to the antibiotic-resistant strains emerged to date. METHODS: We successfully developed the compounds of Rice-fluid derived from unpolished, polished, and usually cooked Japanese rice, and investigated their in vitro antibacterial activities by means of the Time-Kill-Curve methods against various species of bacteria including H. pylori strains. RESULTS: All of the compounds revealed keen bactericidal activities against H. pylori, followed by Streptococcus pneumoniae and Campylobacter jejuni strains, but failed to affect the viability of other bacterial species investigated including staphylococci, enterococci, Pseudomonas aeruginosa, and other gram-negative rods belonging to the family Enterobacteraceae. The bactericidal activities were demonstrated to be time- and concentration-dependent. CONCLUSIONS: The compounds of Rice-fluid are considered to be potentially new and safe therapeutic regimens against H. pylori infections. The mechanism of their bactericidal activities against H. pylori strains remains to be elucidated.

Applicability assessment of ceramic microbeads coated with hydroxyapatite-binding silver/titanium dioxide ceramic composite earthplus™ to the eradication of Legionella in rainwater storage tanks for household use.

Water environments appear to be the habitats of Legionella species. Legionellosis is considered as a preventable illness because bacterial reservoirs can be controlled and removed. Roof-harvested rainwater has attracted significant attention not only as a groundwater recharge but also as a potential alternative source of nonpotable water. We successfully developed ceramic microbeads coated with hydroxyapatite-binding silver/titanium dioxide ceramic composite earthplus™ using the thermal spraying method. The ceramic microbeads were demonstrated to have bactericidal activities against not only Legionella but also coliform and heterotrophic bacteria. Immersing the ceramic microbeads in household rainwater storage tanks was demonstrated to yield the favorable eradication of Legionella organisms. Not only rapid-acting but also long-lasting bactericidal activities of the ceramic microbead were exhibited against Legionella pneumophila. However, time-dependent attenuation of the bactericidal activities against Legionella were also noted in the sustainability appraisal experiment. Therefore, the problems to be overcome surely remain in constantly managing the Legionella-pollution by means of immersing the ceramic microbeads. The results of our investigation apparently indicate that the earthplus™-coated ceramic microbeads would become the favorable tool for Legionella measures in household rainwater storage tanks, which may become the natural reservoir for Legionella species. Our investigation would justify further research and data collection to obtain more reliable procedures to microbiologically regulate the Legionella in rainwater storage tanks.

Physical and genetic map of Enterococcus faecium ATCC19434 and demonstration of intra- and interspecific genomic diversity in enterococci.

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.

Bath water contamination with Legionella and nontuberculous mycobacteria in 24-hour home baths, hot springs, and public bathhouses of Nagano Prefecture, Japan.

Bath water samples were collected from 116 hot springs, 197 public bathhouses, and 38 24-hour home baths in Nagano Prefecture, Japan, during the period of April 2009 to November 2011, for determining the presence and extent of contamination with Legionella and nontuberculous mycobacteria. Cultures positive for Legionella were observed in 123 of the 3,314 bath water samples examined. The distribution and abundance of Legionella and/or combined contamination with Legionella and nontuberculous mycobacteria were investigated to clarify the contamination levels. The abundance of Legionella was demonstrated to correlate considerably with the levels of combined contamination with Legionella and nontuberculous mycobacteria. Legionella spp. were obtained from 61% of the water samples from 24-hour home baths, but only from 3% of the samples from public bathhouses and hot springs. This is despite the fact that a few outbreaks of Legionnaires' disease in Nagano Prefecture as well as other regions of Japan have been traced to bath water contamination. The comparatively higher rate of contamination of the 24-hour home baths is a matter of concern. It is therefore advisable to routinely implement good maintenance of the water basins, particularly of the 24-hour home baths.

First isolation of oleate-dependent Enterococcus faecalis small-colony variants from the umbilical exudate of a paediatric patient with omphalitis.

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children's Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK-semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as ß-lactams.

First Isolation of carbon dioxide-dependent Proteus mirabilis from an uncomplicated cystitis patient with Sjögren's syndrome.

An uncomplicated cystitis caused by CO2-dependent Proteus mirabilis was observed in a 64-year-old Japanese female patient with Sjögren's syndrome in the Aomori Kyoritsu Hospital, Aomori, Japan. The initial P. mirabilis isolate came from a midstream urine specimen containing large numbers of Gram-negative, rod-shaped organisms that failed to grow on both Drigalski agar and sheep blood agar incubated in ambient air. The organism did grow when the urine was cultured overnight on blood agar under anaerobic conditions. Hence, we believed that the organism was an anaerobe. Further investigation revealed that the isolate grew on sheep blood agar along with swarming when the atmospheric CO2 concentrations were increased to 5%. Initially, we failed to characterize or identify the P. mirabilis isolate or determine its antimicrobial susceptibilities using the MicroScan WalkAway-40 System because the isolate did not grow in the system. However, the isolate was subsequently identified as P. mirabilis based on its morphological, cultural, and biochemical properties by using the commercially available kit systems, Quick ID-GN and ID-Test EB-20. This identification of the isolate was confirmed by sequencing the 16S rRNA gene of the organism. To our knowledge, this is the first clinical isolation of capnophilic P. mirabilis.

Isolation and molecular characterization of catalase-negative Staphylococcus aureus from sputum of a patient with aspiration pneumonia.

Staphylococcus aureus produces various virulence factors. The catalase enzyme, in particular, is considered to be involved in oxidative stress resistance, and catalase activity is an important criterion for differentiating staphylococci from streptococci. In this report, we describe the catalase-negative S. aureus strain SH3064, which was isolated from the sputum of a patient with aspiration pneumonia. To evaluate the causes of the lack of catalase activity in S. aureus SH3064, we analyzed the sequence of katA gene encoding the catalase enzyme in this strain. We amplified the complete sequence of katA gene of S. aureus SH3064 by polymerase chain reaction using 2 sets of primers. The katA sequence showed 99.6% sequence identity (1512/1518 bp) with that of S. aureus ATCC 12600. We detected 2 mutations in the katA gene from S. aureus SH3064, an A217T substitution leading to a threonine 73-to-serine substitution and a single-base pair deletion (c.637delG) resulting in a frameshift mutation. The lack of catalase activity in this strain was attributed to the shift of the nucleotide reading frame.

Bactericidal activities of woven cotton and nonwoven polypropylene fabrics coated with hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite "Earth-plus".

BACKGROUND: Bacteria from the hospital environment, including linens and curtains, are often responsible for hospital-associated infections. The aim of the present study was to evaluate the bactericidal effects of fabrics coated with the hydroxyapatite-binding silver/titanium dioxide ceramic nanocomposite "Earth-plus". METHODS: Bactericidal activities of woven and nonwoven fabrics coated with Earth-plus were investigated by the time-kill curve method using nine bacterial strains, including three Staphylococcus aureus, three Escherichia coli, and three Pseudomonas aeruginosa strains. RESULTS: The numbers of viable S. aureus and E. coli cells on both fabrics coated with Earth-plus decreased to below 2 log(10) colony-forming units/mL in six hours and reached the detection limit in 18 hours. Viable cell counts of P. aeruginosa on both fabrics coated with Earth-plus could not be detected after 3-6 hours. Viable cells on woven fabrics showed a more rapid decline than those on nonwoven fabrics. Bacterial cell counts of the nine strains on fabrics without Earth-plus failed to decrease even after 18 hours. CONCLUSION: Woven cotton and nonwoven polypropylene fabrics were shown to have excellent antibacterial potential. The woven fabric was more bactericidal than the nonwoven fabric.