In vitro effect of an antimicrobial combination therapy without colistin and tigecycline for CPE and non-CPE.

INTRODUCTION: This study aimed to investigate the in vitro effects of a combination of antimicrobials other than colistin (CL) and tigecycline (TGC) on carbapenem-resistant Enterobacteriaceae (CRE). METHODS: We used 72 CRE strains including 65 carbapenemase-producing Enterobacteriaceae (CPE) that produce IMP-1, IMP-6, NDM, KPC, and OXA-48-like carbapenemases; and 7 carbapenemase-nonproducing Enterobacteriaceae (non-CPE) strains. These strains were assessed using antimicrobial susceptibility testing, breakpoint checkerboard (BC) plate method, and kill curve experiment to determine the effect of the combination therapy. RESULTS: NDM, KPC, and OXA-48-like carbapenemase-producers showed higher MICs of carbapenem and aminoglycosides, and lower MICs of minocycline, compared to non-CPE and IMP-1/-6-producers. The results of the BC plate method suggested that the suitability of combinations of antimicrobials differ depending on the type of carbapenemases. Killing curve experiments demonstrated bactericidal or bacteriostatic action of the combination of antimicrobials even in sub-MIC concentrations of drugs. Our results suggest that the most effective antimicrobial combinations for each carbapenemase-producers are as follows; IMP-1 (tobramycin + tazobactam/piperacillin), IMP-6 (gentamicin + meropenem), NDM (minocycline + biapenem), KPC (arbekacin + doripenem) and OXA-48-like (minocycline + imipenem). CONCLUSION: These results suggest that the combination of antimicrobials other than CL and TGC may be another candidate for the treatments of CPE infections, even though we have to choose effective antimicrobial combinations depending on the type of carbapenemase.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for clinical microbiology in 2014: General view of the pathogens' antibacterial susceptibility.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2014. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between January 2014 and April 2015 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standards Institute. Susceptibility testing was evaluated in 1534 strains (335 Staphylococcus aureus, 264 Streptococcus pneumoniae, 29 Streptococcus pyogenes, 281 Haemophilus influenzae, 164 Moraxella catarrhalis, 207 Klebsiella pneumoniae, and 254 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 43.6%, and those of penicillin-susceptible S. pneumoniae was 100%. Among H. influenzae, 8.2% of them were found to be ß-lactamase-producing ampicillin-resistant strains, and 49.1% to be ß-lactamase-non-producing ampicillin-resistant strains. Extended spectrum ß-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo ß-lactamase were 9.2% and 0.4%, respectively.

High Prevalence of Antimicrobial Resistance in Gram-Negative Bacteria Isolated from Clinical Settings in Egypt: Recalling for Judicious Use of Conventional Antimicrobials in Developing Nations.

This study was designed to investigate, at the molecular level, the antimicrobial resistance mechanisms of different antimicrobial resistance genes, including, extended-spectrum ß-lactamases, AmpC ß-lactamases, class 1 and 2 integrons, and plasmid-mediated quinolone resistance genes of Gram-negative bacteria isolated from clinical settings in Egypt. A total of 126 nonduplicate Gram-negative isolates were recovered from different clinical samples taken from hospitalized patients in Egypt in 2014. Antimicrobial susceptibility testing showed that, 93.6% (118/126) of the isolates had a multidrug-resistant phenotype. Interestingly, we reported a high level of antimicrobial resistance nearly for all tested antibiotics; to our knowledge, this is the first report from Egypt indicating very high level of antibiotic resistance in Egypt. Polymerase chain reaction screening and DNA sequencing revealed that, 75.4% (95/126) of the isolates harbored at least one extended-spectrum ß-lactamase-encoding gene, with blaCTX-M being the most prevalent (65.9%), followed by blaSHV (46.8%). The AmpC ß-lactamase, blaCMY, was detected in 7.1% (9/126) of bacterial isolates, with blaCMY-42 being the most prevalent. Class 1 integrons were detected in 50.8% (64/126) of the isolates, and class 2 integrons were detected in 2.4% (3/126) of the isolates. The plasmid-mediated quinolone resistance gene, qnr, was detected in 58.7% (74/126) of the tested isolates, with qnrS being the most prevalent. Several antimicrobial resistance determinants were identified in Egypt for the first time, such as SHV-27, SHV-28, SHV-33, SHV-63, SHV-71, SHV-82, SHV-142, CMY-42, CMY-6, and the new CMY-72 like. This study highlights the importance of the conscious use of conventional antimicrobials to overcome the multidrug resistance problem.

Serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae strains isolated in Japan after introduction of the routine immunization program.

Pneumococcal vaccines have reduced the incidences of Streptococcus pneumoniae infections among children and adults, but a relative increase in the prevalence of non-vaccine serotypes has been reported. To follow the changing epidemiology of pneumococcal diseases, capsular serotyping and antimicrobial susceptibility testing was performed on 534 pneumococcal isolates obtained from a hospital in Japan after routine immunization was launched, between October 2014 and May 2016. Serotype distributions and antimicrobial susceptibilities were evaluated among the total patient population, and were compared by age and sample groups and by serotype group, respectively. Serotypes targeted by the 13-valent pneumococcal conjugate vaccine (PCV13) were identified in 14.6%, 44.5%, and 40.2% of the samples from the <5, 5-64, and ≥65 year age groups, respectively. The 23-valent pneumococcal polysaccharide vaccine serotypes (PPSV23) were identified in 42.4%, 68.2%, and 63.1% of the samples, respectively; whereas non-PCV13 serotypes or non-PPSV serotypes (NVT) comprised 46.8% of all isolates. Among NVT, strain 35B was the most frequently isolated, followed by 15A, particularly in sputum samples collected from children <5 years old. Meanwhile, serotype 3, which is targeted by the PCV13 and PPSV23, was the most prevalent among patients aged ≥65 and 5-64 years. Antimicrobial susceptibility testing revealed that 88.9% and 81.0% of serotype 35B was non-susceptible to penicillin and meropenem, respectively, and 89.4% of 15A was non-susceptible to penicillin. Our data suggest rapid effects of pneumococcal vaccines and progression of serotype replacement. Besides invasive potential, the increased prevalence of non-vaccine serotypes with highly non-susceptible to penicillin was a concern. Continuous monitoring of pneumococcal serotypes and antimicrobial susceptibility is necessary for developing optimal preventive strategies.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2010: General view of the pathogens' antibacterial susceptibility.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from patients in Japan, was conducted by Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases and Japanese Society for Clinical Microbiology in 2010. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period from January and April 2010 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical and Laboratory Standard Institutes using maximum 45 antibacterial agents. Susceptibility testing was evaluable with 954 strains (206 Staphylococcus aureus, 189 Streptococcus pneumoniae, 4 Streptococcus pyogenes, 182 Haemophilus influenzae, 74 Moraxella catarrhalis, 139 Klebsiella pneumoniae and 160 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was as high as 50.5%, and those of penicillin-intermediate and -resistant S. pneumoniae were 1.1% and 0.0%, respectively. Among H. influenzae, 17.6% of them were found to be ß-lactamase-non-producing ampicillin (ABPC)-intermediately resistant, 33.5% to be ß-lactamase-non-producing ABPC-resistant and 11.0% to be ß-lactamase-producing ABPC-resistant strains. Extended spectrum ß-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo ß-lactamase were 2.9% and 0.6%, respectively. Continuous national surveillance of antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

Antibiotic susceptibility survey of blood-borne MRSA isolates in Japan from 2008 through 2011.

We conducted an antibiotic susceptibility survey of 830 blood-borne methicillin resistant Staphylococcus aureus collected from nationwide hospitals in Japan over a three-year period from January 2008 through May 2011. Antibiotic susceptibility was judged according to the criteria recommended by the Clinical Laboratory Standard Institute. Over 99% of the MRSA showed to be susceptible to teicoplanin, linezolid, sulfamethoxazole/trimethoprim and vancomycin, and over 97% of them were susceptible to daptomycin, arbekacin and rifampin. The majority of the MRSA strains showed resistant to minocycline, meropenem, imipenem, clindamycin, ciprofloxacin, cefoxitin, and oxacillin in the rates of 56.6, 72.9, 73.7, 78.7, 89.0, 99.5, and 99.9%, respectively. Among the MRSA strains, 72 showed reduced susceptibility to vancomycin, including 8 strains (0.96%) of vancomycin-intermediate S. aureus (VISA), 54 (6.51%) of heterogeneous vancomycin-intermediate S. aureus (hVISA), and 55 (5.63%) of ß-lactam antibiotics-induced vancomycin resistant S. aureus (BIVR). Unexpectedly, among the 54 hVISA and 55 BIVR, 45 isolates (83.3% and 81.8%, respectively) showed both hVISA and BIVR phenotypes. A new trend of vancomycin resistance found in this study was that VISA strains were still prevalent among the bacteremic specimens. The high rates of the hVISA/BIVR two-phenotypic vancomycin resistance, and the prevalence of VISA in the bloodborne MRSA call attention in the MRSA epidemiology in Japan.

Fulminant colitis from Clostridium difficile infection, the epidemic strain ribotype 027, in Japan.

In December 2012, a 32-year-old woman with no previous medical history and no previous antibiotic treatment had a fever and diarrhea 2 days after a cesarean section in which cefazolin was used as a prophylactic antimicrobial agent. She was transferred to our hospital 5 days after the cesarean for severe colitis. A rapid test of stool for Clostridium difficile toxin A and B was positive. Although oral vancomycin (0.5-2.0 g/day) and intravenous immunoglobulin (5 g/day) were administered after her transfer, 7 days after admission emergency exploratory surgery was performed because of poor response to therapy. Bowel perforation was noted and a temporary colostomy was created without colectomy. Vancomycin (2.0 g/day) was administered via the colostomy, in addition to a vancomycin enema (2.0 g/day), oral metronidazole (1500 mg/day), and oral vancomycin (2.0 g/day). Three days after the operation, linezolid (1200 mg/day IV) was added. She was treated with antibiotics against C. difficile for a total of 18 days after the operation. The same strain was not isolated from other patients in the same ward. Microbiological analysis of the isolate revealed housekeeping gene (tpi), toxin A gene (tcdA), toxin B gene (tcdB), and binary toxin gene (cdtA and cdtB). DNA sequencing of tcdC revealed a base 117 deletion and contained an 18-bp tcdC deletion. PCR ribotyping showed ribotype 027 patterns. The MIC of moxifloxacin was >32 µg/ml, indicating resistance to fluoroquinolones. This isolate was considered as the epidemic strain. Our case of fulminant colitis is apparently the first case involving the epidemic strain ribotype 027 in Japan.

Effectiveness of antibiotic combination therapy as evaluated by the Break-point Checkerboard Plate method for multidrug-resistant Pseudomonas aeruginosa in clinical use.

Multidrug-resistant Pseudomonas aeruginosa (MDRP) strains are defined as having resistance to the following 3 groups of antibiotics: carbapenems, aminoglycosides, and fluoroquinolones. Antibiotic combinations have demonstrated increased activity in vitro compared with a single agent. As an in vitro method of determining the combination activity of antibiotics, the Break-point Checkerboard Plate (BC-plate) can be used routinely in clinical microbiology laboratories. We evaluated the effectiveness of the BC-plate for MDRP infections in clinical settings. We retrospectively selected cases of MDRP infection treated with combination therapy of antibiotics in Tokyo Medical University Hospital (1015 beds), Tokyo, Japan, from November 2010 to October 2012. A total of 28 MDRP strains were clinically isolated from 28 patients during the study period. This study design is a case series of MDRP infection. Six infections among the 28 patients were treated based on the results of the BC-plate assay, and the 6 strains tested positive for MBL. One patient had pneumonia, 3 had urinary tract infections, 1 had vertebral osteomyelitis, and 1 had nasal abscess. The combination of aztreonam with amikacin demonstrated the most frequently recognized in vitro effect (5 patients). Next, aztreonam with ciprofloxacin and piperacillin with amikacin revealed equivalent in vitro effects (3 patients, respectively). The clinical cure rate was 83.3% (5/6 patients). Antibiotic combination therapy based on the results of the BC-plate assay might indicate the effective therapy against MDRP infection in clinical settings.

Inhibitory effects of lactoferrin on biofilm formation in clinical isolates of Pseudomonas aeruginosa.

Lactoferrin, a multifunctional protein with antimicrobial activity, is a component of the innate immune system. It may possibly prevent clinical isolates of Pseudomonas aeruginosa from developing biofilm, but this hypothesis is yet to be widely accepted. We evaluated the in vitro effects of lactoferrin on biofilm formation by various clinical isolates of P. aeruginosa using a modified method of the microtiter plate biofilm assay. Lactoferrin significantly inhibited biofilm formation in these isolates. The effect was the most marked at 2 mg/ml, which suggested that an optimal concentration of lactoferrin might exist. Lactoferrin inhibited biofilm formation in eight of nine clinical isolates after 1 day of incubation; however, the inhibitory effects were maintained until 7 days of incubation in only two of those eight strains. Suppression of biofilm formation may be caused by a mechanism that is independent of the bactericidal effects of lactoferrin because the number of viable bacteria was not influenced by lactoferrin under the experimental conditions. Supplementation of lactoferrin to preformed biofilm demonstrated a reduction in biofilm, which suggests that lactoferrin may have a destructive effect on biofilm. Pretreatment with ferric chloride partially restored biofilm formation, suggesting an iron-chelating action may be involved in the inhibitory mechanism of lactoferrin. These results suggest that lactoferrin provides inhibitory effects on biofilm formation in many clinical isolates of P. aeruginosa and that it may also have destructive effects on preformed biofilm, but further research using multiple clinical strains should be undertaken to clarify if those effects are universal.

Efficacy of bacteriophage therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice.

We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P<0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-alpha, interleukin-1beta [IL-1beta], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa.

The in vitro and in vivo antibacterial characterization of vancomycin and linezolid against vancomycin-susceptible and -resistant enterococci.

The present study was designed to compare in vitro antibacterial activities of linezolid and vancomycin against vancomycin-susceptible Enterococcus faecalis (VSEF) and vancomycin-resistant enterococci (VRE) isolated in Japan with those of quinupristin-dalfopristin, teicoplanin and minocycline, and the in vitro short time bactericidal activity and the in vivo activities of linezolid and vancomycin against vancomycin-susceptible and -resistant E. faecalis. The MIC90s of linezolid, quinupristin-dalfopristin, vancomycin, teicoplanin and minocycline for VSEF and VRE were both 2 mg/L, both 2 mg/L, 2 and >128 mg/L, 0.25 and >128 mg/L, and both 32 mg/L, respectively. The efficacy of linezolid for mice with bacteraemia caused by VSEF was similar to that of vancomycin, but the elimination ratio of viable organisms from the blood of mice treated with vancomycin was significantly higher than in linezolid-treated and untreated mice at 2 h post-administration, and those of the two groups at 4 and 6 h were significantly higher than in untreated mice. Moreover, linezolid was highly active in mice with bacteraemia caused by vancomycin-resistant E. faecalis because this drug had potent in vitro activity against the organisms. Our results indicate that linezolid is suitable for the treatment of VRE and VSEF bacteraemia, and vancomycin is suitable for VSEF bacteraemia.