The effect of gut microbiota and probiotic organisms on the properties of extended spectrum beta-lactamase producing and carbapenem resistant Enterobacteriaceae including growth, beta-lactamase activity and gene transmissibility.

The gut microbiota may play a pivotal role in controlling the antimicrobial resistant (AMR) organisms although the evidences are limited. We investigated the effects of gut microbiota on the growth of AMR organisms, ß-lactamases activity and transmissibility of antimicrobial resistant properties of the extended spectrum ß-lactamase (ESBL)-producing Escherichia coli and carbapenem-resistant Enterobacteriaceae. CTX-M-15-positive, ESBL-producing E. coli and carbapenem resistant Enterobacteriaceae, Bacteroides fragilis, Bifidobacterium longum, Clostridium butyricum, Clostridioides difficile, Clostridium perfringens, Enterococcus faecium, Lactobacillus plantarum and probiotic strain of C. butyricum MIYAIRI 588 were used in this study. The growth of AMR organisms was suppressed by the supernatant of C. butyricum, C. difficile, C. perfringens, E. faecium and L. plantarum in a dose dependent manner but not by that of B. fragilis and B. longum. The ß-lactamase activity produced by E. coli was reduced by the presence of culture supernatant of certain gut microbiota during stationary phase of E. coli. Importantly, C. butyricum MIYAIRI 588 culture supernatant suppressed the transcription of blaCTX-M gene during growth phase of E. coli. The conjugation assay showed the reduction of transmissibility of antibiotic resistant gene by gut microbiota. These findings suggest that certain gut microbiota affect the antibiotic resistant activities of AMR organisms. Further studies are needed to identify the specific mechanism(s) of these actions between AMR organisms and gut microbiota.

First confirmed case of spondylodiscitis with epidural abscess caused by Parvimonas micra.

Parvimonas micra was renamed species as within Gram-positive anaerobic cocci and rarely causes severe infections in healthy people. We report the first confirmed case of spondylodiscitis with epidural abscess caused by P. micra in a healthy women. The patient has a pain in low back and anterior left thigh. Magnetic resonance imaging and computed tomography detected the affected lesion at the L2 and L3 vertebral bodies. All isolates from the surgical and needle biopsy specimens were identified as P. micra by 16S rRNA and MALDI-TOF. In this case, P. micra showed high sensitivity to antimicrobial therapy. She was successfully treated with debridement and sulbactam/ampicillin, followed by oral metronidazole for a total of 10 weeks. The causative microorganisms of spondylodiscitis are not often identified, especially anaerobic bacteria tend to be underestimated. On the other hand, antimicrobial therapy for spondylodiscitis is usually prolonged. Accordingly, we emphasize the importance of performing accurate identification including anaerobic bacteria.

A survey conducted immediately after the 2011 Great East Japan Earthquake: evaluation of infectious risks associated with sanitary conditions in evacuation centers.

In cooperation with the Miyagi prefectural government, we conducted a survey of the management of sanitation at evacuation centers and the health of the evacuees by visiting 324 evacuation centers at two weeks after the 2011 Great East Japan Earthquake. The facilities often used as evacuation centers were community centers (36%), schools (32.7%) and Nursing homes (10.2%). It was more difficult to maintain a distance of at least 1 m between evacuees at the evacuation centers with a larger number of residents. At evacuation centers where the water supply was not restored, hygienic handling of food and the hand hygiene of the cooks were less than adequate. Among evacuation centers with ≤50 evacuees, there was a significant difference in the prevalence rate of digestive symptoms between the centers with and without persons in charge of health matters (0.3% vs. 2.1%, respectively, p < 0.001). The following three factors had an important influence on the level of sanitation at evacuation centers and the health of evacuees: 1) the size of the evacuation center, 2) the status of the water supply, and 3) the allocation of persons in charge of health matters. Given that adjusting the number of evacuees to fit the size of the evacuation center and prompt restoration of the water supply are difficult to achieve immediately after an earthquake, promptly placing persons in charge of health matters at evacuation centers is a practicable and effective measure, and allocation of at least one such person per 50 evacuees is desirable.

High frequency of Acinetobacter soli among Acinetobacter isolates causing bacteremia at a tertiary hospital in Japan.

Acinetobacter baumannii is generally the most frequently isolated Acinetobacter species. Sequence analysis techniques allow reliable identification of Acinetobacter isolates at the species level. Forty-eight clinical isolates of Acinetobacter spp. were obtained from blood cultures at Tohoku University Hospital. These isolates were identified at the species level by partial sequencing of the RNA polymerase ß-subunit (rpoB), 16S rRNA, and gyrB genes. Then further characterization was done by using the PCR for detection of OXA-type ß-lactamase gene clusters, metallo-ß-lactamases, and carO genes. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were also performed. The most frequent isolate was Acinetobacter soli (27.1%). Six of the 13 A. soli isolates were carbapenem nonsusceptible, and all of these isolates produced IMP-1. PFGE revealed that the 13 A. soli isolates were divided into 8 clusters. This study demonstrated that A. soli accounted for a high proportion of Acinetobacter isolates causing bacteremia at a Japanese tertiary hospital. Non-A. baumannii species were identified more frequently than A. baumannii and carbapenem-nonsusceptible isolates were found among the non-A. baumannii strains. These results emphasize the importance of performing epidemiological investigations of Acinetobacter species.

Tuberculosis exposure among evacuees at a shelter after earthquake, Japan, 2011.

Tuberculosis was diagnosed in a person who had stayed in a shelter after the 2011 Great East Japan Earthquake. A contact investigation showed that the prevalence of latent tuberculosis infection among other evacuees at the shelter was 20%. Our report underscores the importance of tuberculosis prevention and control after natural disasters.

Molecular characteristics of extended-spectrum ß-lactamases in clinical isolates from Escherichia coli at a Japanese tertiary hospital.

The prevalence of ESBL has been increasing worldwide. In this study, we investigated the molecular characteristics of ESBL among clinical isolates of Escherichia coli from a Japanese tertiary hospital. A total of 71 consecutive and nonduplicate clinical isolates of ESBL-positive E. coli collected at Tohoku University Hospital between January 2008 and March 2011 were studied. The antimicrobial susceptibility profile of these strains was determined. PCR and sequencing were performed to identify genes for ß-lactamase (bla(TEM), bla(SHV), bla(OXA-1-like), and bla(CTX-M)) and plasmid-mediated quinolone resistance determinants (PMQR). The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Of the 71 strains, 68 were positive for CTX-M, 28 were positive for TEM, four were positive for OXA-1, and one was positive for SHV. Sequencing revealed that CTX-M-14 was the most prevalent (31/71), followed by CTX-M-27 (21/71) and then CTX-M-15 (9/71). Of the 28 TEM-positive strains, one was TEM-10 and the rest were TEM-1. One SHV-positive strain was SHV-12. The 21 CTX-M-27-producing isolates were divided into 14 unique PFGE types, while the 9 CTX-M-15 producers were divided into 8 types. Based on MLST, 9 CTX-M-14 procedures, 19 CTX-M-27 procedures, and 8 CTX-M-15 producers belonged to ST131. Thirty-five (94.6%) of the 37 ST131 E. coli strains showed resistance to levofloxacin, which was a higher rate than among non-ST131 strains (63.6%). Among ESBL-producing isolates, one, two, and six possessed qnrB, qnrS, qepA, and aac(6')-Ib-cr, respectively. Of the 6 isolates with aac(6')-Ib-cr, 4 carried the CTX-M-15 gene. Our data suggest that CTX-M-15-producing E. coli ST131 has emerged as a worldwide pandemic clone, while CTX-M-27 (a variant of CTX-M-14) is also spreading among E. coli ST131 in Japan.

Characteristics of infectious diseases in hospitalized patients during the early phase after the 2011 great East Japan earthquake: pneumonia as a significant reason for hospital care.

BACKGROUND: Natural catastrophes increase infectious disease morbidity rates. On March 11, 2011, a 9.0-magnitude earthquake and associated Pacific coast tsunami struck East Japan. The aim of this study was to investigate the characteristics of patients with infectious diseases who needed hospitalization after this disaster. METHODS: We searched the medical records of 1,577 patients admitted to Tohoku University Hospital in the Sendai area within 1 month (March 11, 2011-April 11, 2011) after the disaster. We examined (1) changes in the rates of hospitalizations for infectious diseases over time and (2) the variety of infectious diseases. RESULTS: The number of hospitalized patients with infectious diseases increased after the fi rst week to double that during the same period in 2010. Pneumonia comprised 43% of cases, and 12% consisted of skin and subcutaneous tissue infection, including tetanus. Pneumonia was prevalent in elderly patients (median age, 78 years) with low levels of serum albumin and comorbid conditions, including brain and nervous system disorders. Sputum cultures contained Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae , known pathogens of community-acquired pneumonia in Japan. In addition, 20.5% of patients had positive results for urinary pneumococcal antigen. CONCLUSIONS: Among hospitalized patients, infectious diseases were significantly increased after the disaster compared with the same period in 2010, with pneumonia being prominent. The analyses suggest that taking appropriate measures for infectious diseases, including pneumonia, may be useful for disaster preparedness and medical response in the future.

Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan.

The incidence of extended-spectrum ß-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC ß-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.

Molecular epidemiology of carbapenem-non-susceptible Acinetobacter baumannii in Japan.

OBJECTIVES: Acinetobacter baumannii presents a clinical challenge when it is non-susceptible to carbapenems. The prevalence of carbapenem-non-susceptible A. baumannii in Japan is unclear, as previous studies have been limited in scope. We investigated the spread of carbapenem-non-susceptible A. baumannii in Japan and performed a comparison with findings from overseas. METHODS: A total of 305 non-duplicate clinical isolates of Acinetobacter spp. from 176 medical facilities in all geographical regions of Japan were tested for susceptibility to antimicrobial agents by the agar dilution method. Isolates with MICs of imipenem ≥ 4 mg/L underwent PCR analysis of OXA-type ß-lactamase gene clusters and metallo-ß-lactamase genes. These isolates were further analysed by sequencing of OXA-type ß-lactamases and by multilocus sequence typing (MLST). RESULTS: Fifty-five of the 305 clinical isolates had MICs of imipenem ≥ 4 mg/L. The OXA-51-like carbapenemase gene was detected in 52 of these 55 isolates. Within the OXA-51-like gene cluster, OXA-66 was found in 43 (82.7%) of the 52 isolates. MLST identified the following sequence types (STs): ST74, ST76, ST92, ST106, ST188 and ST195 in 2 (3.8%), 2 (3.8%), 40 (76.9%), 5 (9.6%), 2 (3.8%) and 1 (1.9%) of the isolates, respectively. In particular, ST92 was found in 31 (91.2%) of the 34 A. baumannii isolates with MICs of imipenem ≥ 16 mg/L. CONCLUSIONS: This is the first report on the molecular epidemiology of A. baumannii with MICs of imipenem ≥ 4 mg/L in Japan. OXA-66 and ST92 were dominant among these isolates.

Post-tsunami outbreaks of influenza in evacuation centers in Miyagi Prefecture, Japan.

We describe 2 post-tsunami outbreaks of influenza A in evacuation centers in Miyagi Prefecture, Japan, in 2011. Although containment of the outbreak was challenging in the evacuation settings, prompt implementation of a systemic approach with a bundle of control measures was important to control the influenza outbreaks.

Bactericidal effects of antimicrobial agents on epithelial cell-associated Pseudomonas aeruginosa.

It is not clear whether antipseudomonal agents can kill cell-associated bacteria within a short time. Madin-Darby canine kidney (MDCK) and A549 cells were infected with Pseudomonas aeruginosa ATCC 27853 and PAO1 and the bactericidal activity of ceftazidime, imipenem, meropenem, gentamicin, and ciprofloxacin against the organisms was investigated. In both MDCK and A549 cells, ß-lactams could not kill epithelial cell-associated bacteria within 2 h. Gentamicin at concentrations ≤32 µg/ml killed more than 99% of epithelial cell-associated bacteria. Ciprofloxacin at 0.5 µg/ml killed more than 99.9% of MDCK cell-associated bacteria. Ciprofloxacin has the strongest and most rapid bactericidal activity against epithelial cell-associated bacteria, which may be explained by the combination of potent in-vitro bactericidal activity and high penetration ability into epithelial cells.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2009].

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.

High prevalence of extended-spectrum ß-lactamases and qnr determinants in Citrobacter species from Japan: dissemination of CTX-M-2.

OBJECTIVES: Extended-spectrum ß-lactamases (ESBLs) have become a problem among AmpC-producing Enterobacteriaceae and the emergence of concomitant quinolone resistance in ß-lactamase-producing isolates poses a global threat. In this study we investigated the prevalence and regional variation of ESBLs in Japanese clinical isolates of Citrobacter spp. and analysed plasmid-mediated quinolone resistance (PMQR) determinants in ESBL-producing Citrobacter spp. METHODS: A total of 348 clinical isolates of Citrobacter spp. collected throughout Japan were studied. Screening and the boronic acid disc test were performed to detect ESBLs in Citrobacter spp. with chromosomal AmpC ß-lactamases. PCR and sequencing were done to identify ESBL and PMQR genes. For ESBL-producing Citrobacter spp., PFGE was performed using the SfiI restriction enzyme. RESULTS: The number of ESBL-producing isolates confirmed phenotypically was 67 (19.3%). The prevalence of ESBL-producing Citrobacter koseri was significantly higher (32.1%) than that of ESBL-producing Citrobacter freundii (4.6%) (P < 0.01). Moreover, the prevalence of ESBLs was notably higher among C. koseri from southern Japan (60.0%). CTX-M-2 was predominant in C. koseri. Of the ESBL-producing C. koseri analysed, 23.2% possessed PMQR determinants, and there was a significant association between qnrB4 and bla(SHV-12). The 57 ESBL-producing Citrobacter spp. possessing bla(CTX-M), bla(SHV) or bla(TEM) were divided into 18 unique PFGE types. CONCLUSIONS: This is the first report about the prevalence of PMQR determinants among ESBL-producing Citrobacter spp. from Japan. Our data suggest that ESBLs and PMQR determinants are spreading among C. koseri in Japan.

The increase in surface CXCR4 expression on lung extravascular neutrophils and its effects on neutrophils during endotoxin-induced lung injury.

Inflammatory stimuli, such as a microbes or lipopolysaccharides, induce a rapid release of neutrophils from the bone marrow and promote neutrophil migration into inflamed sites to promote host defense. However, an excess accumulation and retention of neutrophils in inflamed tissue can cause severe tissue injuries in the later stages of inflammation. Recent studies have reported that both CXCL12 levels in injured lungs and its receptor, CXCR4, on accumulated neutrophils in injured lungs, increased; furthermore, these studies showed that the CXCL12/CXCR4 signaling pathway participated in neutrophil accumulation in the later stages of lipopolysaccharide (LPS)-induced lung injury. However, the mechanisms underlying this increase in surface CXCR4 expression in neutrophils remain unclear. In this study, we found that surface CXCR4 expression increased in extravascular, but not intravascular, neutrophils in the lungs of LPS-induced lung injury model mice. Furthermore, ex vivo studies revealed that CXCL12 acted not only as a chemoattractant, but also as a suppressor of cell death for the lung neutrophils expressing CXCR4. Sulfatide, one of the native ligands for L-selectin, induced the increase of surface CXCR4 expression on isolated circulating neutrophils, suggesting that the activation of L-selectin may be involved in the increase in surface CXCR4. Our findings show that surface CXCR4 levels on neutrophils increase after extravasation into injured lungs, possibly through the activation of L-selectin. The CXCL12/CXCR4 signaling pathway plays an important role in the modulation of neutrophil activity during acute lung injury, not only by promoting chemotaxis but also by suppressing cell death.

Emergence of fluoroquinolone-resistant Streptococcus pyogenes in Japan by a point mutation leading to a new amino acid substitution.

OBJECTIVES: Streptococcus pyogenes causes various diseases in humans. While the prevalence of fluoroquinolone-resistant S. pyogenes isolates has been increasing since 2000 in the USA and Europe, it has remained very low in Japan. We isolated a fluoroquinolone-resistant S. pyogenes strain and analysed its genetics. METHODS: TU-296, a strain of S. pyogenes resistant to levofloxacin (MIC 16 mg/L), was isolated from the throat of a patient in their thirties with pharyngitis in autumn 2007. We carried out susceptibility tests for various antimicrobial agents and PCR analysis of the genes gyrA, gyrB, parC and parE in the quinolone resistance-determining region, followed by sequencing of the PCR products to find mutation(s) and the resulting amino acid substitution(s). We then sequenced the PCR product of the emm gene and determined the emm genotype. RESULTS: S. pyogenes TU-296 was found to have the following mutations and amino acid substitutions: adenine 476 to cytosine in gyrA and cytosine 367 to thymine in parC, resulting in Glu-85→Ala in GyrA and Ser-79→Phe in ParC. The genotype of the isolate was emm11. CONCLUSIONS: Amino acid substitutions in fluoroquinolone-resistant S. pyogenes have already been reported from Europe and the USA, including Ser-81→Phe or Tyr and Met-99→Leu in GyrA, as well as Ser-79→Phe, Tyr or Ala and others in ParC. Numerous point mutations were found in parC and parE of S. pyogenes TU-296. In addition, a new amino acid substitution was detected (Glu-85→Ala in GyrA). To our knowledge, there have been no previous reports of this substitution in a clinical isolate of S. pyogenes.

Methicillin resistant Staphylococcus aureus in a Japanese community hospital: 5-year experience.

Methicillin resistant Staphylococcus aureus (MRSA) is exceptionally critical to infection treatment and control in the health-care setting. MRSA has been detected at high levels in Japan, and the frequency of MRSA infection must be ascertained to provide a baseline with which to assess various infection control efforts. We studied MRSA infection rate at a general hospital in Japan in all 65,135 inpatients of Sendai Kousei Hospital from January 2004 to December 2008. MRSA's prevalence among strains of S. aureus and the rate of MRSA detection were studied. Identification of MRSA infection is according to the laboratory-based ward liaison surveillance. The minimal inhibitory concentrations (MICs) of vancomycin, teicoplanin, and arbekacin for the various isolates were determined. During the period studied, there were 621 MRSA-positive patients. MRSA prevalence among strains of S. aureus was 45.5% (621/1,365). The rate of MRSA detection in inpatients was 0.953/100 inpatients. Of the 621 patients from whom MRSA was isolated, 51 (8.2%) had an MRSA infection (MRSA infection rate 0.078/100 inpatients). MRSA was often detected from the respiratory tract, but this seldom led to infection, since many of those affected were merely carriers. MICs against MRSA was 0.5-4 µg/ml for vancomycin, 0.5-16 µg/ml for teicoplanin, and 0.5 to >16 µg/ml for arbekacin, with no tendency for tolerance observed for these drugs. Findings suggest that whereas MRSA remains prevalent, there is a low incidence of infection in a general hospital in Japan.

Monolayer culture systems with respiratory epithelial cells for evaluation of bacterial invasiveness.

Pseudomonas (P.) aeruginosa is a major opportunistic pathogen especially in immunocompromised patients. To evaluate the invasiveness of respiratory pathogens, we developed monolayer culture systems and examined the degree of invasion by P. aeruginosa and invasive Salmonella (S.) typhimurium strains using human respiratory cell lines: A549 (derived from lung cancer), BEAS-2B (normal bronchial epithelium), and Calu-3 (pleural effusion of a patient with adenocarcinoma of the lung). Cells were seeded into filter units containing 0.33 cm(2) filter membranes with 3.0 microm pores, and were incubated at 37 degrees C under 5% CO(2) for 4-10 days. By monitoring the trans-monolayer electrical resistance (TER), we judged that BEAS-2B cells (TER values: 436.2 +/- 16.8 to 628.8 +/- 66.3 Omega cm(2)) and Calu-3 cells (TER values: 490.5 +/- 25.2 to 547.8 +/- 21.6 Omega cm(2)) formed monolayers with tight junctions, but not A549 cells. On day 8 of culture, monolayer cultures were infected with bacteria, and the number of microorganisms penetrating into the basolateral medium was counted. Wild-type P. aeruginosa PAO1 (PAO1 WT) and S. typhimurium SL1344 were detected in the basolateral medium of BEAS-2B monolayer system by 3 h after inoculation, while only P. aeruginosa PAO1 WT was detected in the basolateral medium of Calu-3 monolayer, indicating poor invasiveness of S. typhimurium SL1344 in the Calu-3 system. These findings suggest that BEAS-2B or Calu-3 monolayer system could be useful for evaluating the invasiveness of respiratory pathogens. Because of the difference in bacterial invasiveness, we may need to choose a suitable cell system for each target pathogen.