Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Characterization and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from chicken and pork.

The prevalence and antimicrobial resistance (AMR) profile were investigated in Campylobacter jejuni and Campylobacter coli in chicken and pork in Fukuoka, Japan in 2019. Their AMR profiles were compared with those of C. jejuni and C. coli strains isolated in 2013. A total of 53 chicken and 14 pork samples were collected from different supermarkets in Fukuoka in 2019. Campylobacter spp. were isolated by conventional method and characterized by PCR and MALDI-TOF MS. Among 53 chicken samples tested in 2019, 24.5% and 5.7% were positive for C. jejuni and C. coli, respectively, and three (21.4%) of 14 pork samples were positive for C. coli, but not C. jejuni. From the positive samples, 13 and six strains of C. jejuni and C. coli were isolated, respectively. Antimicrobial susceptibility test against 12 different antimicrobials were performed on 48 isolates (43 C. jejuni and five C. coli) from chicken in 2013 and 19 isolates (13 C. jejuni from chicken, three C. coli from chicken and three C. coli from pork) in 2019 using the disk diffusion method. All the C. jejuni and C. coli isolated in 2013 and 2019 were highly resistant to cefazolin and sulfamethoxazole/trimethoprim. Among the C. jejuni isolates from chickens, 25.6% of 2013 isolates were resistant to nalidixic acid, ciprofloxacin, and levofloxacin, and 7% to ampicillin and minocycline, while 30.8% of the isolates were resistant to minocycline, 23.1% to nalidixic acid, ciprofloxacin, and levofloxacin, and 15.4% to ampicillin in 2019. Among the C. coli isolates, 80% of isolates from chickens in 2013, and 33.3% from chicken and 100% from pork in 2019 were resistant to nalidixic acid, ciprofloxacin, and levofloxacin. The frequency of multi-drug resistant (MDR) C. jejuni and C. coli strains from chickens in 2019 were 30.8% and 33.3%, respectively, which were lower than those isolated in 2013 (37.2% and 100%, respectively). One C. jejuni and two C. coli isolates from 2013 were resistant to six antibiotics. However, two C. jejuni and one C. coli isolate from chickens in 2019 were resistant to seven and five antibiotics, respectively. All the C. coli isolates from pork in 2019 were resistant to five antibiotics. The high frequency of AMR strains in C. coli isolates from pork suggests that appropriate use of antimicrobials is required in swine husbandry.

Characterization and Application of Lytic Bacteriophages against Campylobacter jejuni Isolated from Poultry in Japan.

　The aim was to isolate Campylobacter jejuni-specific lytic phages from meats on the market in Japan. These phages were effectively isolated from 13 of 15 (86.7%) retail chicken meat samples (skin and liver) by the enrichment method using Preston Campylobacter Selective Enrichment Broth and 10 host Campylobacter strains. Among the 26 phage isolates, 14 were extracted by means of C. jejuni L26 as a host strain. Phage PHC10 showed the broadest lytic spectrum: active against 67.4% of the 46 C. jejuni strains tested. The other phage isolates showed different lytic spectra. Because phages PHC5, PHC10, PHC19, PHC22, and PHC25 possess an icosahedral head and a contracted tail, they seem to be members of the Myoviridae family. Effects of 19 phage isolates on viability of C. jejuni were investigated. These phages reduced viable counts of C. jejuni by 1-3 log after 6-12 h of incubation at 42℃ as compared to the initial counts. The C. jejuni L26 was found to be suitable as a host because of the wide hosting range. The phages isolated in this study seem to be promising biocontrol agents against C. jejuni in food.

[Bursting risk of capped PET bottles with leftover contents caused by yeast growth].

Accidents caused by the bursting of capped PET bottles with leftover contents have increased in Japan. Therefore, the bursting risk of capped PET bottles with leftover contents was investigated. The ratios of distension, after 10 days at 25 degrees C, of capped PET bottles with leftover orange juice were approx. 70% in the case of drinking while eating Kimuchi, and approx. 6% in the case of drinking but not eating. Among 58 distended bottles, none burst spontaneously, but 4 bottles (6.9%) burst upon physical shock (dropping onto a concrete floor from 1.5 m height). The greatest distensions of capped PET bottles were observed with leftover orange juice, apple juice and soured milk after 10 days at 25 degrees C. No distensions were observed in bottles containing leftover sports drink and green tea under the same conditions. Identified organisms from distended bottles were almost all Candida spp. When those Candida spp. strains were inoculated individually into PET bottles half full of orange, apple or sour milk, all bottles were distended after 7 days at 25 degrees C. From the above results, it appears that there is a risk that capped PET bottles with leftover contents, especially sour fruit juice or soured milk, may burst owing to high gas pressure caused by yeast growth during storage at room temperature for over 1 week.

Increase in resistance to extended-spectrum cephalosporins in Salmonella isolated from retail chicken products in Japan.

Extended-spectrum ß-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996-2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the bla(CMY), bla(CTX-M), bla(TEM), and bla(SHV) resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (bla(CMY-2)), and nine ESBL-producing isolates harboring bla(CTX-M) (n = 4, consisting of two bla(CTX-M-2) and two bla(CTX-M-15 genes)), bla(TEM) (n = 4, consisting of one bla(TEM-20) and three bla(TEM-52) genes), and bla(SHV) (n = 1, bla(SHV-12)). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing bla(CMY-2) in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated bla(CMY-2) in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health.