Bayesian reconstruction of a vancomycin-resistant Enterococcus transmission route using epidemiologic data and genomic variants from whole genome sequencing.

BACKGROUND: Outbreaks of vancomycin-resistant enterococcus (VRE) are a serious problem in hospitals. Inferring the transmission route is an important factor to institute appropriate infection control measures; however, the methodology has not been fully established. AIM: To reconstruct and evaluate the transmission model using sequence variants extracted from whole genome sequencing (WGS) data and epidemiological information from patients involved in a VRE outbreak. METHODS: During a VRE outbreak in our hospital, 23 samples were collected from patients and environmental surfaces and analysed using WGS. By combining genome alignment information with patient epidemiological data, the VRE transmission route was reconstructed using a Bayesian approach. With the transmission model, evaluation and further analyses were performed to identify risk factors that contributed to the outbreak. FINDINGS: All VREs were identified as Enterococcus faecium belonging to sequence type 17, which consisted of two VRE genotypes: vanA (N = 8, including one environmental sample) and vanB (N = 15). The reconstruction model using the Bayesian approach showed the transmission direction with posterior probability and revealed transmission through an environmental surface. In addition, some cases acting as VRE spreaders were identified, which can interfere with appropriate infection control. Vancomycin administration was identified as a significant risk factor for spreaders. CONCLUSION: A Bayesian approach for transmission route reconstruction using epidemiologic data and genomic variants from WGS can be applied in actual VRE outbreaks. This may contribute to the design and implementation of effective infection control measures.

Successful control of an outbreak of GES-5 extended-spectrum ß-lactamase-producing Pseudomonas aeruginosa in a long-term care facility in Japan.

BACKGROUND: Little is known about multidrug-resistant Pseudomonas aeruginosa (MDRP) outbreaks in long-term care facilities (LTCFs). AIM: To describe an MDRP outbreak in an LTCF and to clarify risk factors for MDRP acquisition. METHODS: Patients who were positive for MDRP at an LTCF from January 2013 to January 2014 were analysed. A descriptive analysis, a case-control study, and a microbiological analysis were performed. FINDINGS: A total of 23 MDRP cases were identified, 16 of which were confirmed in sputum samples. Healthcare workers were observed violating hand hygiene procedures when performing oral, wound, and genital care. Nasogastric tube and oxygen mask use was associated with MDRP acquisition in the respiratory tract, which might have been confounded by poor hand hygiene. Sharing unhygienic devices, such as portable oral suction devices for oral care, and washing bottles and ointments for wound and genital care with inadequate disinfection could explain the transmission of MDRP in some cases. Isolates from 11 patients were found to be indistinguishable or closely related by pulsed-field gel electrophoresis and harbouring the blaGES-5 gene. Subsequent enhanced infection control measures were supported by nearby hospitals and a local public health centre. No additional cases were identified for a year after the last case occurred in January 2014. CONCLUSION: An outbreak of MDRP with an antimicrobial resistance gene, blaGES-5, occurred in a Japanese LTCF. It was successfully controlled by enhanced infection control measures, which neighbouring hospitals and a local public health centre supported.

Increase in paratyphoid fever cases in Japanese travellers returning from Cambodia in 2013.

In 2013, an unusual increase of paratyphoid fever cases in travellers returning from Cambodia was reported in Japan. From December 2012 to September 2013, 18 cases of Salmonella Paratyphi A infection were identified. Microbiological analyses revealed that most isolates had the same clonal identity, although the epidemiological link between these cases remains unclear. It was inferred that the outbreak was caused by a common and persistent source in Cambodia that was likely to have continued during 2014. The information of surveillance and laboratory data from cases arising in travellers from countries with limited surveillance systems should be timely shared with the country of origin.

Dependence of fructooligosaccharide content on activity of fructooligosaccharide-metabolizing enzymes in yacon (Smallanthus sonchifolius) tuberous roots during storage.

Tuberous roots of yacon (Smallanthus sonchifolius) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy benefits. However, we have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting even under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8 degrees C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (beta-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on sucrose, both invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FEH acting on short FOSs such as 1-kestose (GF(2)) and 1-nystose (GF(3)) was higher than that of 1-FFT. These results suggest that the continuous decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand, FOSs with a DP of >or= 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct 1-FEH activity was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connected with the 1-FEH activity.

Hop bract polyphenols reduced three-day dental plaque regrowth.

Previous research has shown the inhibitory effects of hop bract polyphenols (HBP) on cariogenic streptococci in vitro, but their effects in humans have not been investigated. This double-blind, crossover clinical study tested the hypothesis that HBP delivered in a mouthrinse suppresses plaque regrowth in humans. Twenty-nine healthy male volunteers had all plaque removed, and refrained from all oral hygiene for 3 days, except for rinsing with a mouthrinse containing 0.1% HBP or a placebo. The results showed that the mean amount of plaque assessed by the Patient Hygiene Performance score after the volunteers used the HBP mouthrinse was significantly less than that after they used the placebo (p < 0.001). The number of mutans streptococci in the plaque samples after volunteers used the HBP mouthrinse was significantly lower than that after they used the placebo (p < 0.05). These findings suggested that HBP, delivered in a mouthrinse, successfully reduced dental plaque regrowth in humans.

Modification of IkappaBalpha by taurine bromamine inhibits tumor necrosis factor alpha-induced NF-kappaB activation.

OBJECTIVE AND DESIGN: Eosinophils play a prominent role in the pathogenesis of various common human allergic diseases, including asthma. Taurine chloramine (TauCl) and taurine bromamine (TauBr) are products of activated neutrophils and eosinophils. TauCl has strong anti-inflammatory properties. However, much less is known about TauBr. The aim of this study was to compare the anti-inflammatory capacity and membrane permeability of TauBr to those of TauCl. MATERIALS AND METHODS: Jurkat cells (T-lymphocytes) and YJ cells (myeloid-committed eosinophils) were used throughout this in vitro study. Tumor necrosis factor (TNFalpha) was employed for activation of the cells. Degradation of the cytosolic NF-kappaB inhibitor protein (IkappaBalpha) was studied by Western blot analysis. Assessment of NF-kappaB DNA binding activity was performed by an electrophoretic mobility shift assay (EMSA). RESULTS: TauBr inhibited degradation of IkappaBalpha and TNFalpha-induced NF-kappaB activation. TauBr exerted an anti-inflammatory effect by a similar process to that of TauCl. TauBr administered extracellularly in phosphate buffered saline (PBS) shifted the IkappaBalpha band at a relatively low concentration of 50 muM. In addition, TauBr was membrane-permeable as demonstrated by the inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CONCLUSIONS: TauBr was found to be highly membrane-permeable. TauBr might be generated both extracellularly and intracellularly by eosinophils at inflammatory sites in allergic disease and play an anti-inflammatory role.

Comparison of Neisseria gonorrhoeae isolates from the genital tract and pharynx of two gonorrhea patients.

Neisseria gonorrhoeae was isolated simultaneously from urethral and pharyngeal specimens of two gonorrhea patients. The pair of isolates from one of the two patients were identical in auxotype, pulsed-field gel electrophoresis pattern, and antimicrobial susceptibility, which indicated that both sites in that patient were infected with the same strain of N. gonorrhoeae. However, the two isolates from the other patient had different properties, indicating that the two sites in the second patient were infected with different strains. The gonococcal infections in these patients failed to respond to initial treatment with sparfloxacin or sulbactam/ampicillin, because the causative strains of N. gonorrhoeae were resistant to the respective antibiotics. Variable patterns and routes of gonococcal infection have recently been discovered in individual patients, suggesting that specimens for bacterial isolation should be taken not just from one site but from various sites that might be infected. This method may contribute to the successful treatment and epidemiological investigation of gonococcal infections.

Association between proline-requiring auxotype and fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Japan.

We examined the association between auxotype and fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Fukuoka, Japan, and investigated whether the prevalence of fluoroquinolone-resistant N. gonorrhoeae isolates was caused by the dissemination of the same clone in the community. We examined the antimicrobial susceptibility of 294 N. gonorrhoeae, isolates obtained during three different periods in Fukuoka, Japan, and 89 isolates of N. gonorrhoeae, classified by the presence of amino-acid substitutions in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC proteins, to various agents, and we examined the auxotypes of the isolates. In 22 isolates with amino-acid substitutions within QRDRs in GyrA and ParC, pulsed-field gel electrophoresis analysis was performed. The proportion of fluoroquinolone-resistant isolates (ciprofloxacin, minimum inhibitory concentration [MIC] > or = 1 microg/ml) in 1998 (23.9%) was significantly higher than that in 1992-1993 (0%). The proportion of proline-requiring isolates increased significantly, from 4.4% in 1992-1993 to 54.5% in 1998. The ciprofloxacin MIC90 for the proline-requiring isolates were 32- and 128-fold, respectively, higher than those for the prototrophic isolates and the arginine-requiring isolates. The proportion of isolates with amino-acid substitutions within the QRDRs in GyrA and ParC in the proline-requiring group (55.5%) was significantly higher than that in the prototrophic group (0%). Of the 22 isolates with amino-acid substitutions within the QRDRs in GyrA and ParC, 16 showed the same pulsed-field gel electrophoresis pattern. These results suggest that a close association exists between the increase in the proline-requiring isolates and the increase in the fluoroquinolone-resistant isolates in the gonococci isolated in Fukuoka, and that the prevalence of fluoroquinolone-resistant N. gonorrhoeae isolates with GyrA and ParC substitutions may be mainly caused by the dissemination of a single clone in the community.

Antimicrobial activity of gemifloxacin (SB-265805), a newer fluoroquinolone, against clinical isolates of Neisseria gonorrhoeae, including fluoroquinolone-resistant isolates.

Antimicrobial activity of gemifloxacin (SB-265805), a newly developed fluoroquinolone, to Japanese isolates of Neisseria gonorrhoeae was compared with those of various fluoroquinolones, including norfloxacin, ciprofloxacin, tosufloxacin, levofloxacin, sparfloxacin, and trovafloxacin. Among the fluoroquinolones tested, gemifloxacin was most active against N. gonorrhoeae isolates. The MIC90 values of gemifloxacin for 94 N. gonorrhoeae isolated from 1992 through 1993 and 100 isolated from 1996 through 1997 were 0.03 and 0.125 microg/ml, respectively. On the other hand, MIC90 values of the other fluoroquinolone for the 1992-1993 isolates and the 1996-1997 isolates ranged from 0.125 to 2 microg/ml and from 0.5 to 8 microg/ml, respectively. Gemifloxacin was also the most potent fluoroquinolone against 31 ciprofloxacin-resistant isolates with the ciprofloxacin MIC of 1 to 16 microg/ml, for which the gemifloxacin MIC50 and MIC90 values were 0.25 and 2 microg/ml, respectively. Moreover, the activity of gemifloxacin against fluoroquinolone-resistant gonococcal isolates containing multiple amino acid substitutions in both GyrA and ParC proteins was superior to those of the other compounds.

Identification of a taurine transport inhibitory substance in sesame seeds.

An ethanol extract from sesame seeds inhibited the taurine uptake in human intestinal epithelial Caco-2 cells. The uptake of such alpha-amino acids as leucine and glutamic acid was not inhibited by the extract, indicating that this inhibition is specific to the taurine uptake. The unknown inhibitor in the sesame extract was purifled by reversed-phase HPLC by monitoring the inhibitory effect on taurine uptake. The isolated substance was identified as lysophosphatidylcholine, linoleoyl (Lyso-PC), by NMR and MS analysis. Lyso-PC inhibited the taurine uptake in a dose-dependent manner with an IC50 value of approximately 200 microM. Although Lyso-PC is known to be a surface active and cell lytic compound, neither damage nor loss of integrity of the Caco2 cell monolayer was apparent after treating with 200 microM Lyso-PC. Inhibition was observed by incubating cells with Lyso-PC for only 1 min prior to the uptake experiments. These results suggest the direct effect of Lyso-PC on the cell membrane to be the main mechanism for this inhibition. Lyso-PC may play a role in the regulation of certain intestinal transporters.

Comparison of gyrA and parC mutations and resistance levels among fluoroquinolone-resistant isolates and laboratory-derived mutants of oral streptococci.

Laboratory-derived fluoroquinolone-resistant mutants were obtained by serial passage of Streptococcus sanguis and Streptococcus anginosus isolates on agar containing increasing concentrations of old and new fluoroquinolones, ofloxacin and DU-6859a, respectively. Sequencing of an S. sanguis isolate exposed to DU-6859a showed that resistance was associated with two mutations in the quinolone resistance determining region (QRDR) of the gyrA gene (Ser83-->Phe; Glu87-->Lys), and with a mutation in the parC gene (Ser79-->Ile). However, different mutations in the gyrA gene (Ser83-->Tyr) and parC gene (Ser79-->Phe) were found in a S. sanguis isolate exposed to ofloxacin. A fluoroquinolone-resistant isolate, QR-95101, from a dental infection, had a single mutation in the gyrA gene (Ser83-->Phe) and in the parC gene (Ser79-->Phe). Two fluoroquinolone-resistant mutants, QS-701OFm and QS-701DUm, were obtained from S. anginosus QS-701, by exposure to ofloxacin and DU-6859a, respectively. These mutants showed a common substitution at codon 83 (Ser-->Phe) in the gyrA gene but had different substitutions at codon 87 (QS-701OFm, Glu-->Gln; QS-701DUm, Glu-->Lys). They also had different substitutions at codons 79 and 135 in the parC gene (QS-701OFm, Ser79-->Leu but no change at Glu135; QS-701DUm, Ser79-->Ile and Glu135-->Gln). The resistance levels of the DU-6859a-selected resistant S. sanguis mutant QS-951DUm to DU-6859a, ofloxacin, ciprofloxacin and norfloxacin were higher than those of the ofloxacin-selected resistant mutant QS-951OFm. However, ampicillin susceptibilities of these mutants were not different from the parental strains. In S. anginosus, the DU-6859a-selected fluoroquinolone-resistant mutant QS-701DUm was resistant to all the fluoroquinolones tested, while the ofloxacin-selected mutant QS-701OFm was resistant to three fluoroquinolones, but not DU-6859a. The results indicate that different fluoroquinolones select distinct mutations in the QRDR of the gyrA and parC genes in oral streptococci. The gyrA or parC mutation in oral streptococci may determine the levels of fluoroquinolone resistance.

High prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in female commercial sex workers in Japan.

Our objectives were to explore the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium in Japanese female commercial sex workers (CSWs), in comparison with pregnant women as controls. A high-risk group of 174 female CSWs and 90 asymptomatic pregnant women were enrolled in this study. Detection of C. trachomatis, N. gonorrhoeae, and M. genitalium on the endocervix of the women was performed mainly by using polymerase chain reaction (PCR)-based assays. The prevalence rates of C. trachomatis, N. gonorrhoeae, and M. genitalium were 19.0%, 32.8%, and 12.6%, respectively, in the CSWs, compared with 5.6%, 0%, and 1.1% respectively, in the pregnant women. These results suggest a high prevalence of C. trachomatis, N. gonorrhoeae, and M. genitalium in Japanese CSWs. We conclude that continued close monitoring of the prevalence of C. trachomatis, N. gonorrhoeae, and M. genitalium infection in CSWs is important for preventing the dissemination of these microorganisms, and that further investigation of M. genitalium as a sexually transmitted pathogen in women is needed.

[Study of diarrhea-inducing strains of Escherichia coli mainly isolated in Kanto area between June and September 1996].

A total of 21,609 faecal specimens obtained from patients with diarrhea mainly in Kanto area between June and September 1996 were investigated to identify the causative pathogens for diarrhea. One-hundred fifty-seven strains of Escherichia coli of 29 different O-serotypes were isolated as the causative pathogens, which were previously recognized to induce severe abdominal cramps and diarrhea. Of these, 114 strains, in which the possibility of enterohemorrhagic E. coli due to their O-serotypes was predicted, were examined for the producibility of Vero toxins. Twenty-six (76.5%) of the 34 strains of E. coli O157 produced the Vero toxins, and other 8 strains were the non-producers. Twenty of the 26 producers produced both VT1 and VT2, whereas the other 6 strains produced VT2 only. Furthermore, 4 strains of E. coli O26, and 1 strain each of E. coli O125 and O126 produced Vero toxins. Thirty-two of the 114 strains, isolated from the patients with diarrhea and selected as the enterohemorrhagic E. coli according to the specific O-serotypes, were actually confirmed produce Vero toxins. Thirty-four strains of E. coli O157 tested were susceptible to all antibiotics such as ampicillin, doxycylin, levofloxacin, fosfomycin, chloramphenicol and polymyxin B, and no strains resistant to levofloxacin, polymyxin B and fosfomycin were found.

[Drug-resistance patterns and biological properties of MRSA isolated from different geographical areas of Japan].

One hundred isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected from hospitals located in different geographical areas of Japan were used in the present study. The susceptibilities of the strains to gentamicin (GM), erythromycin (EM), tetracycline (TC) and ofloxacin (OFLX) were determined and classified into twelve groups according to their differences in the patterns of the resistance to the four drugs. Of the 100 strains tested, 75 belonging to the main four groups were investigated for the relationships between their patterns of the drug-resistance and biological properties such as coagulase, enterotoxin and phage types, TSST-1 and beta-lactamase producibilities, and percentages of the strains carrying plasmids of large size (> or = 20,000 bp). The main four patterns of drug resistance of the 75 strains were as follows: the first group (33 strains resistant to GM, EM, TC and OFLX), the second group (15 strains resistant to EM, TC and OFLX), the third group (11 strains resistant to GM, EM and OFLX) and the fourth group (16 strains resistant to EM and OFLX) and the remaining 25 strains were divided into further groups. A considerable number of the strains in the third group differed markedly in some biological properties from those in the other groups; coagulase typing (III type-50%: the other groups-II type), enterotoxin typing (64%-non-production: the other groups-C type), TSST-1 producibility (36%-production: the other groups-76 to 88%-production) and phage typing (55%-non-typable: the other groups-80 to 97%-non-typable). In beta-lactamase and plasmids of large size, the first group consisted of the strains with beta-lactamase producibility at the ratio of 50:50, but the other groups consisted of most of the strains with the beta-lactamase producibility. Most of the strains belonging to the second and fourth groups carried large size of plasmids, but 36 and 46% of those belonging to the first and third groups did not carry them. On the other hand, our results of the susceptibility test for the 100 strains showed that a considerable number of strains were susceptible to GM (35%) and TC (42%) at therapeutically significant concentrations.

[Effects of azithromycin on fecal flora of healthy adult volunteers].

New macrolide antibiotic, azithromycin (AZM), was administered to six healthy male volunteers and its effects on their intestinal microflora were investigated. Each volunteer was given 500 mg of AZM orally, once a day for 3 consecutive days. Stool samples were obtained from them prior to the medication and 1, 7, 14 and 28 days after the third day the medication. A slight decrease in the total aerobic bacterial count was observed. Also, several species of anaerobic bacteria showed slight decreases through the 14th day post medication. Individual variances were observed, however. A marked decrease in the number of Bifidobacterium was found for each of the volunteers. Clostridium difficile was detected from one of the volunteers on the 28th day post medication without diarrhea related symptoms.