Differences in flaA gene sequences, swimming motility, and biofilm forming ability between clinical and environmental isolates of Aeromonas species.

The flagellin A gene (flaA) sequences, swimming motility, and biofilm forming ability were investigated in order to reveal the genetic and functional differences of flagella between clinical and environmental isolates of Aeromonas species. Twenty-eight clinical and 48 environmental strains of Aeromonas species isolated in Okinawa Prefecture of Japan were used in this study. The full-length flaA genes of these strains were sequenced and aligned, and a phylogenetic tree was constructed. In addition, swimming motility and biofilm forming ability were evaluated by conventional methods. Aeromonas veronii biovar sobria and A. hydrophila clearly divided into clinical and environmental strain clusters in the flaA phylogenetic classification, and the six and 13 specific amino acids respectively, of FlaA of both species were different in clinical and environmental strains. Furthermore, the flaA size of the clinical strain of A. veronii bv. sobria was mainly 909, 924, and 939 bp, and the size of A. hydrophila was 909 bp. The swimming motility of clinical isolates of both species was lower than the environmental isolates; however, the biofilm forming ability of the clinical isolates was high. Thus, the clinical isolates of A. veronii bv. sobria and A. hydrophila had different genetic and functional characteristics of flagellin than the environmental isolates. The characteristics of flagellin could serve as indicators to distinguish between clinical and environmental isolates of the both species. It may contribute to diagnosis of these diseases and the monitoring of clinical strain invasion into the natural environment.

Occurrence of Carriage of Multidrug Resistant Enterobacteriaceae among Pregnant Women in the Primary Health Center and Hospital Setting in Surabaya, Indonesia.

Objectives: The incidence of healthy individuals carrying multidrug resistant Enterobacteriaceae, including extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E), especially extended-spectrum ß-lactamase producing Escherichia coli (ESBL-EC) and extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP), is increasing worldwide. Although ESBL-E causes early or late onset of neonatal sepsis, the prevalence of ESBL-E carriage among pregnant women in Indonesia is not clear. In the present study, we compared the occurrence of carriage of ESBL-E among pregnant women in a primary health center (PHC) versus two hospitals. Materials and Methods: We collected rectal swab samples from 200 pregnant women who visited a PHC or were admitted to two hospitals in Surabaya, Indonesia from July to October 2018. The ESBL-E strains were isolated from the samples and phenotypically and genotypically analyzed. Results: ESBL-E strains were isolated from 25 (24.8%) pregnant women who visited the PHC and 49 (49.5%) pregnant women who were admitted to the hospitals. The rate of ESBL-E carriage of pregnant women in the hospitals was significantly higher than that in the PHC. Among the 74 isolated ESBL-E strains, ESBL-EC was most frequently isolated (62 strains), followed by ESBL-KP (12 strains). In addition, blaCTX-M-15 was the most frequent ESBL gene type of the isolated ESBL-E strains. Conclusions: Our results revealed the high occurrence of ESBL-E carriage in pregnant women, especially those who were admitted to the hospitals.

Analysis of the upstream genetic structures of the ISEcp1-blaCTX-M transposition units in Escherichia coli isolates carrying blaCTX-M obtained from the Indonesian and Vietnamese communities.

Extended spectrum ß-lactamase (ESBL)-producing Escherichia coli have been found in healthy individuals in Indonesia and Vietnam. The ISEcp1-blaCTX-M transposition unit of ESBL-producing bacterial isolates has been considered responsible for the production of CTX-M type ESBL and it is important for the dissemination of blaCTX-M . This study aimed to characterize the upstream genetic structure (UGS) of E. coli isolates possessing blaCTX-M-1 group and/or blaCTX-M-9 group genes obtained from healthy individuals in Indonesia and Vietnam. A total of 501 CTX-M type ESBL-producing E. coli isolates possessing blaCTX-M-1 group and/or blaCTX-M-9 group genes were obtained from healthy individuals of the two countries in 2018. The UGSs of the ISEcp1-blaCTX-M transposition unit of the 501 ESBL-producing E. coli isolates were amplified by barcode-adaptor-ligation-mediated PCR and analyzed using the Nanopore sequencer. The obtained sequence information was used to classify the UGSs of the ISEcp1-blaCTX-M transposition unit. From the 501 ESBL-producing E. coli isolates, 502 UGSs were obtained, which were classified into 85 UGS types based on the sequence. ISEcp1 of 359 (71.5%) of the 502 UGSs was disrupted by gene insertion, and ISEcp1-blaCTX-M transposition unit of most (87.1%) of the determined UGSs was confirmed as plasmidic. Only 6 (7.1%) of the 85 UGS types were common to both countries. Our results indicated that many different UGSs of ISEcp1-blaCTX-M transposition units were detected in Indonesia and Vietnam; hence, we suggest that structurally different kinds of plasmids harboring blaCTX-M were separately distributed in the two countries.

Differential effects of chromosome and plasmid blaCTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection.

OBJECTIVES: To compare antibiotic susceptibilities between chromosomal and plasmid blaCTX-M-15 locations in urinary tract infection-causing extended-spectrum ß-lactamases-producing Escherichia coli blaCTX-M-15 isolated in Indonesia. METHODS: A total of 84 strains identified as extended-spectrum ß-lactamases-producing E. coli were isolated from patients with urinary tract infection in Indonesia in 2015. Antimicrobial susceptibility tests were performed on these strains using 18 antibiotics, and extended-spectrum ß-lactamase bla genes were detected by polymerase chain reaction. Gene localization of blaCTX-M-15 -positive strains was confirmed by Southern blot hybridization, and epidemiological typing was conducted using multilocus sequence typing. RESULTS: Of 54 strains harboring the blaCTX-M-15 gene, 27 showed localization on chromosome, 20 on plasmid, and seven on chromosome and plasmid. Most multilocus sequence typing sequence types of the 27 strains with chromosomal blaCTX-M-15 were ST405 (25.9%) and ST131 (22.2%) strains, whereas the 20 strains with plasmid-blaCTX-M-15 were mostly ST410 (55.0%). CONCLUSIONS: Extended-spectrum ß-lactamases-producing E. coli blaCTX-M-15 with plasmid genes show significantly higher resistant rates against piperacillin-tazobactam but lower resistant rates against chloramphenicol compared to chromosomal strains in Indonesian patients with urinary tract infection. Mechanistic investigations will be necessary to advance our knowledge of antimicrobial resistance in urinary tract infection.

A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-blaCTX-M transposition unit and application of the UGS to classification of bacterial isolates possessing blaCTX-M.

INTRODUCTION: Because blaCTX-M is responsible for resistance of bacteria to the third generation cephalosporins, location of blaCTX-M could be a good indicator for classifying bacterial isolates harboring blaCTX-M in molecular epidemiology. However, determination of blaCTX-M location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of blaCTX-M location. METHODS: Extracted DNA samples obtained from 168 Escherichia coli isolates possessing blaCTX-M were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. RESULTS: Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. CONCLUSIONS: Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-blaCTX-M transposition unit was useful for classification of bacterial isolates harboring blaCTX-M.

High Prevalence of Colistin-Resistant Escherichia coli with Chromosomally Carried mcr-1 in Healthy Residents in Vietnam.

The wide distribution of colistin-resistant bacteria in developing countries has become a common phenomenon. To understand the mechanisms underlying their distribution, we studied the mcr genetic background of colistin-resistant Escherichia coli isolates from the fecal microbiota of healthy human residents from a community in Vietnam with a high prevalence of colistin-resistant E. coli with mcr Fifty-seven colistin-resistant isolates were obtained from 98 residents; one isolate was collected from each individual and analyzed for mcr We found that 36.8% of the isolates carried chromosomal mcr-1 Further, 63.2% and 1.8% of the isolates carried mcr-1 on the plasmid and the plasmid/chromosome, respectively. Whole-genome sequencing of genetically unrelated isolates showed that the majority (6 of 7) of the isolates had the chromosomal mcr-1 in a complete ancestral mcr-1 transposon Tn6330, ISApl1-mcr-1-PAP2-ISApl1, which was inserted at various positions on the chromosomes. In addition, the majority (87.5%) of Tn6330 of mcr-1-carrying plasmids (n = 8) lacked both upstream and downstream ISApl1 transposons. The results obtained in this study indicate that plasmid-to-chromosomal transfer of mcr-1 may have occurred recently in the fecal microbiota of the residents. Additionally, Tn6330 on the chromosome may lose ISApl1 from the transposon during multiplication to gain a more stable mcr-1 state on the chromosome. Stabilization of resistance by the chromosomal incorporation of mcr-1 would be an additional challenge in combating the dissemination of resistant bacteria.IMPORTANCE Elucidation of the mechanism of the wide dissemination of colistin-resistant bacteria in communities of developing countries is an urgent public health issue. In this study, we investigated the genetic background of the colistin resistance gene mcr in E. coli isolates from the fecal microbiota of healthy human residents living in a community in Vietnam with a high prevalence of colistin-resistant E. coli Our study revealed for the first time, a surprisingly high percentage (36.8%) of colistin-resistant E. coli carrying chromosomal mcr-1, the emergence of which may have occurred recently, in the fecal microbiota of the community residents. The mcr-1 transposon on the chromosome may develop into a more stable genotype by the loss of insertion sequences (ISs). Our results are valuable in understanding the mechanism underlying the increasing prevalence of colistin-resistant bacteria within a community.

Characterization of CTX-M-type-extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolated from Indonesian undergraduate medical students of a university in Surabaya, Indonesia.

Enterobacteriaceae isolates producing CTX-M-type extended-spectrum ß-lactamase (ESBL) has been found in hospitalized patients and healthy individuals in communities of the Southeast Asian countries. Medical students might have more risk of ESBL-producing Enterobacteriaceae contagion, because medical students who belong to communities have direct and indirect contacts with workers and patients in healthcare facilities. The aim of this study was to collect information for evaluation of the potential risk of ESBL-producing Enterobacteriaceae contagion in Indonesian undergraduate medical students by characterizing genotypic properties of Escherichia coli isolates-producing CTX-M-type ESBL. A total 141 fecal samples collected from 207 medical students of a university in Surabaya, Indonesia were subjected to PCR, XbaI and S1 nuclease-pulsed-field gel electrophoresis (PFGE), Southern blotting, and sequencing analysis. Eighty-two ESBL-producing Enterobacteriaceae, including 75 E. coli and 7 Klebsiella pneumoniae were isolated from 79 (56.0%) students. Among 75 ESBL-producing E. coli, blaCTX-M-15 was the most prevalent type (44.0%). Although XbaI-PFGE results showed genetic background of the E. coli isolates producing CTX-M-type ESBL were diverse, five clonal spread cases of certain E. coli producing CTX-M-type ESBL isolates were observed among the medical students. Our results suggested that ESBL-producing Enterobacteriaceae might be circulating among the medical students through contaminated environment such as in a university or communities they belonged.

Characterization of blaCTX-M-14 transposition from plasmid to chromosome in Escherichia coli experimental strain.

Mostly, blaCTX-M is found on transferable plasmids as a component of the blaCTX-M transposition unit containing an insertion sequence, ISEcp1, which exists on the upstream region of blaCTX-Ms. Several recent studies conducted in clinical and community settings have reported the presence of chromosomally located blaCTX-M in extended spectrum ß-lactamase (ESBL)-producing bacterial isolates. In this study, we observed the frequency and molecular nature of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome by using an experimental strain of Escherichia coli. We determined 102 different chromosomal transposition sites of blaCTX-M-14 in 126 E. coli isolates following five independent screening procedures. The characterization of the 102 different chromosomal transposition sites of blaCTX-M-14 observed in this study revealed the presence of 5-bp direct repeat (DR) sequences and identical left terminal inverted sequences in 80 E. coli isolates. However, 5'-flanking sequences of the right terminal DR sequences in the 80 E. coli isolates were highly diverse, and consensus sequences of the right terminal inverted repeat sequences were not observed. In case of our E. coli experimental strain, the frequency of the ISEcp1-mediated transposition of blaCTX-M-14 from a plasmid to a chromosome was determined to be 0.51% (SD = 0.37). Collectively, the molecular nature of ISEcp1 could plausibly be a factor contributing to the high detection rates of E. coli possessing chromosomally located blaCTX-M-14 in both clinical and community settings.

A survey of extended-spectrum ß-lactamase-producing Enterobacteriaceae in environmental water in Okinawa Prefecture of Japan and relationship with indicator organisms.

Surveys of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-pE) in stream water and untreated wastewater were carried out in Okinawa Prefecture, Japan. Thirty-six samples of water were collected from 18 streams in Okinawa Prefecture, as well as ten samples of wastewater flowing into four wastewater treatment plants (WWTPs). We investigated bacterial species, Escherichia coli O antigen, ESBL phenotype, ESBL genotype, and pulsed-field gel electrophoresis (PFGE) type of isolates, and total viable count and fecal coliforms as indicator organisms. The relation between indicator organisms and ESBL-pE was also validated using the same samples. A total of 141 ESBL-pE including 107 E. coli, 15 Klebsiella pneumoniae, 2 Proteus mirabilis, and 17 other species was isolated from stream water and wastewater. Of the 141 ESBL-pE, 14.9% and 54.6% were found to be blaCTX-M-15 and blaCTX-M-14-like types, respectively, which have been found in hospital isolates in Okinawa. Two pairs of possibly related patterns according to PFGE criteria were isolated from stream water and wastewater in two districts. When ESBL-pE was significantly isolated, total viable count and fecal coliform boundaries were ≥ 6.0 × 103 CFU/ml and ≥ 4.3 × 102 most probable number/100 ml, respectively. These results suggested that ESBL-pE isolated from stream water is human derived, and that total viable count and fecal coliforms will be useful as indicators for confirming the spread of ESBL-pE to the environment by means of simple hygiene surveys.

Characterisation of chromosomally-located blaCTX-M and its surrounding sequence in CTX-M-type extended-spectrum ß-lactamase-producing Escherichia coli isolates.

OBJECTIVES: Although it has been regarded that the CTX-M-type extended-spectrum ß-lactamase (ESBL) gene blaCTX-M is mainly carried by antimicrobial resistance plasmids, Escherichia coli possessing chromosomally-located blaCTX-M has been reported in previous studies. This study aimed to characterise the genetic structure of the chromosomally-located blaCTX-M transposition unit and its surrounding sequence in ESBL-producing E. coli isolated in a Japanese hospital. METHODS: A total of 81 ESBL-producing E. coli isolates were studied. The existence of chromosomally-located blaCTX-M was confirmed by S1 nuclease-digested pulsed-field gel electrophoresis (PFGE) and Southern blot hybridisation and by sequencing analysis of the PCR-amplified DNA fragments. RESULTS: Chromosomally-located blaCTX-M was confirmed in 22 (27.2%) of the 81 E. coli isolates examined; five and four location types of chromosomally-located blaCTX-M-14 and blaCTX-M-15 were determined, respectively. Among the 22 E. coli isolates, 15 (68.2%) possessed single chromosomally-located blaCTX-M gene, probably due to single transposition of a plasmidic blaCTX-M to the chromosome. In isolate N0057, the blaCTX-M-15 transposition unit was transferred from a plasmid into two different chromosomal regions. In addition, 'recurrent' transposition of already existing chromosomally-located blaCTX-M-14 to another chromosomal region was observed in isolates N0211, N0214, N01127, N1682 and N1753; consequently, these isolates possessed two copies of chromosomally-located blaCTX-M-14. CONCLUSION: Considering that isolates N0211, N0214, N01127, N1682 and N1753 in which the 'recurrent' transposition event occurred were genetically related according to PFGE, these data suggest the possibility of accumulation of blaCTX-M on the chromosome in CTX-M-type ESBL-producing E. coli.

Characterization of CTX-M type ESBL-producing Enterobacteriaceae isolated from asymptomatic healthy individuals who live in a community of the Okinawa prefecture, Japan.

This study was performed to characterize CTX-M type extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2 µg/ml of cefotaxime and incubated at 37 °C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of blaCTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E. coli B2 were E. coli B2-ST131. Three CTX-M type ESBL-producing E. coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E. coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred blaCTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E. coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.

Potential transmission opportunity of CTX-M-producing Escherichia coli on a large-scale chicken farm in Vietnam.

OBJECTIVES: Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli have disseminated worldwide. This study investigated blaCTX-M-positive E. coli on a large-scale Vietnamese chicken farm and analysed whether there was any difference in prevalence and molecular characteristics of blaCTX-M-positive E. coli between the farm and the Vietnamese community. METHODS: Faecal samples were collected from 24 human individuals and 38 chickens from the large-scale chicken farm as well as 51 humans and 36 chickens from the community. All samples were collected between June 2013 and June 2014 in Bavi Province in the Red River Delta region of Vietnam. Molecular characterisation of CTX-M-producing E. coli and genetic relatedness among the isolates were evaluated by conventional typing methods. Antimicrobial susceptibility of the isolates was evaluated by the disk diffusion method. RESULTS: The prevalence of blaCTX-M-positive E. coli was 83.3%, 71.1%, 54.9% and 13.9% in farm workers, farm chickens, community individuals and community backyard chickens, respectively. On average, blaCTX-M-positive E. coli isolates from farm chickens were resistant to 8.3 different antibiotics. The average number of detected aminoglycoside-modifying enzyme genes (3.4 genes) and the detection rate of the plasmid-mediated colistin resistance gene mcr-1 (33.3%) were higher in blaCTX-M-positive E. coli isolated from farm chickens compared with other sampling groups. In addition, two types of indistinguishable pulsed-field gel electrophoresis (PFGE) patterns were observed in six blaCTX-M-65-positive E. coli and three blaCTX-M-55-positive E. coli from farm chickens. CONCLUSIONS: These results suggest a more frequent transmission opportunity of blaCTX-M-positive E. coli on the large-scale Vietnamese chicken farm.

Sanitary evaluation of domestic water supply facilities with storage tanks and detection of Aeromonas, enteric and related bacteria in domestic water facilities in Okinawa Prefecture of Japan.

To provide for temporary restrictions of the public water supply system, storage tanks are commonly installed in the domestic water systems of houses and apartment buildings in Okinawa Prefecture of Japan. To learn more about the sanitary condition and management of these water supply facilities with storage tanks (hereafter called "storage tank water systems") and the extent of bacterial contamination of water from these facilities, we investigated their usage and the existence of Aeromonas, enteric and related bacteria. Verbal interviews concerning the use and management of the storage tank water systems were carried out in each randomly sampled household. A total of 54 water samples were collected for bacteriological and physicochemical examinations. Conventional methods were used for total viable count, fecal coliforms, identification of bacteria such as Aeromonas, Enterobacteriaceae and non-fermentative Gram-negative rods (NF-GNR), and measurement of residual chlorine. On Aeromonas species, tests for putative virulence factor and an identification using 16S rRNA and rpoB genes were also performed. Water from the water storage systems was reported to be consumed directly without boiling in 22 of the 54 houses (40.7%). 31 of the sampled houses had installed water storage tanks of more than 1 cubic meter (m3) per inhabitant, and in 21 of the sampled houses, the tank had never been cleaned. In all samples, the total viable count and fecal coliforms did not exceed quality levels prescribed by Japanese waterworks law. Although the quantity of bacteria detected was not high, 23 NF-GNR, 14 Enterobacteriaceae and 5 Aeromonas were isolated in 42.6%, 7.4% and 3.7% of samples respectively. One isolated A. hydrophila and four A. caviae possessed various putative virulence factors, especially A. hydrophila which had diverse putative pathogenic genes such as aer, hlyA, act, alt, ast, ser, and dam. Many bacteria were isolated when the concentration of residual chlorine was below 0.1 mg/l and the water temperature was above 20 °C. These results suggest that elevated water temperature and mismatch between tank size and water demand lead to loss of residual chlorine in tap water. Therefore, to minimize growth of aquatic bacteria such as Aeromonas spp. and Pseudomonas spp., we recommend that an appropriate size tank and/or volume of stored water is always used, and also suggest installation of some means of reducing water temperature such as shading.

Distribution of Aeromonas species in environmental water used in daily life in Okinawa Prefecture, Japan.

OBJECTIVES: The genus Aeromonas is known to causes diseases such as food poisoning, sepsis, and wound infection. However, the mode of Aeromonas transmission from environment to humans is not clearly understood. To evaluate the health risks of Aeromonas spp. in environmental freshwater, the number, proportion and putative virulence factors of Aeromonas species were investigated in Okinawa Prefecture, Japan. METHODS: Environmental freshwater samples were collected from three dams, two springs and three private wells. Aeromonas strains were identified by the biochemical method and the viable count was calculated. The production of extracellular enzymes and the virulence genes were investigated for possessing putative virulence factors using representative isolates. RESULTS: At least seven species of already-known Aeromonas isolates as well as unidentified Aeromonas spp. with/without arginin dehydrolase (ADH) exist in water at these sites. Aeromonas spp. was found to exist at over 1000 CFU/100 ml in one spring and two wells. A. veronii biovar sobria and A. jandaei were the predominant species in dams, and A. hydrophila and/or A. eucrenophila were predominant in wells. Almost all the sampled Aeromonas species produced protease, gelatinase, lipase, esterase and DNase, but A. caviae, A. caviae-like bacteria, and A. eucrenophila had low hemolytic activity. Most sampled A. hydrophila strains possessed both aerolysin gene (aer) and hemolysin gene (hlyA), but A. caviae and A. eucrenophila strains did not possess either gene. CONCLUSIONS: Since these results indicated that several Aeromonas species having potential pathogenicity exist in environmental water in Okinawa, surveys are recommended as a public health measure.

Current status of extended spectrum ß-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan.

Enterobacteriaceae producing extended spectrum ß-lactamase (ESBL) are distributed worldwide. In this study, 114 ESBL-producing Enterobacteriaceae were isolated by analyzing 1672 clinical isolates of Enterobacteriaceae collected from an Okinawa prefectural hospital in Japan between June 2013 and July 2014. The overall prevalence of ESBL-producing Enterobacteriaceae was 6.8%; the prevalence of different bacterial species among the ESBL-producing isolates was as follows: 11.5% Escherichia coli (90 of 783 isolates), 6.2% Klebsiella pneumoniae (19 of 307 isolates), and 11.1% Proteus mirabilis (5 of 45 isolates). The ESBL types blaCTX-M-1, -3, -15, -2, -14, -27, and mutants of blaSHV-1 were detected. Among them, blaCTX-M-15 (33.3%), blaCTX-M-14 (27.8%) and blaCTX-M-27 (33.3%) were dominant in the E. coli isolates, whereas a blaSHV mutant which possessed four mutations (Tyr7Phe, Leu35Gln, Gly238Ser and Glu240Lys) in the amino acid sequence of SHV-1 dominated in the K. pneumoniae isolates (11 of 19, 57.9%). The pandemic E. coli ST131 clone was found to constitute 3.3% of the overall examined isolates and 62.2% of the ESBL-producing E. coli isolates. Our results suggest that the genetic combination of blaCTX-M, and blaSHV and antibiotics-resistant profile were different from that in other regions such as other areas of Japan, Asia, Europe, and North America, especially in the ESBL-producing K. pneumoniae isolates and in the E. coli B2-O25b-ST131 isolates possessing blaCTX-M-15 (40.7% of the E. coli B2-O25b-ST131 isolates). Taken together, our results indicate that the ESBL-producing Enterobacteriaceae in Okinawa, Japan, might be of a unique nature.

Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection.

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infectivity is thought to play a crucial role in severe dengue disease. It occurs when pre-existing sub-neutralizing anti-DENV antibody (Ab) produced from a primary infection encounters a DENV serotype different from that of the initial infection and forms immune complexes, which enable the efficient infection of Fcγ receptor-bearing cells. However, the exact role played by Abs during a secondary infection of patients remains unknown. We previously obtained a broadly cross-reactive neutralizing IgG1 human monoclonal anti-DENV envelope (E) Ab (HuMAb) D23-1G7C2-IgG1 from a DENV-infected patient; however, D23-1G7C2-IgG1 had ADE activity. With the aim of being able to reduce the ADE activity, we exchanged the Fc region of D23-1G7C2 to generate Abs bearing each of the three other IgG subclasses (IgG2-4). In addition, N297A, a mutation known to reduce the affinity of the IgG1 Fc region for Fcγ receptors, was introduced into D23-1G7C2-IgG1. Swapping D23-1G7C2-IgG1 to IgG2 or IgG4 subclasses reduced ADE activity in FcγRI and FcγRII-bearing THP-1 cells. By contrast, in FcγRII-bearing K562 cells, the change to IgG2 increased ADE activity. Introducing the N297A mutation into D23-1G7C2-IgG1 resulted in a marked reduction in ADE activity in both cell types. Compared to D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A was less protective in IFN-α/ß/γ receptor knockout mice infected with a lethal dose of recombinant chimeric DENV, carrying prME of DENV-2 in Japanese encephalitis virus (80% vs. 40% survival, respectively). These observations provide valuable information regarding the use of recombinant Abs as therapeutics.

Carriage of Escherichia coli Producing CTX-M-Type Extended-Spectrum ß-Lactamase in Healthy Vietnamese Individuals.

Healthy carriage of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli was examined by thrice collecting fecal samples from the same 199 healthy Vietnamese subjects every 6 months. Using pulsed-field gel electrophoresis (PFGE), identical PFGE patterns throughout the three samplings were not observed, although prevalence of E. coli in the subjects was around 50% in the three samplings. Our results suggested a short carriage period of the CTX-M-type ESBL-producing E. coli in healthy Vietnamese subjects.

Characteristics of Extended-Spectrum ß-Lactamase-Producing Escherichia coli in Retail Meats and Shrimp at a Local Market in Vietnam.

BACKGROUND: Contamination of food with multiantibiotic-resistant bacteria, particularly extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, is considered a potential source for the wide dissemination of ESBL-producing bacteria in communities. However, little is known about the extent of contamination of food with ESBL-producing bacteria in Vietnam. OBJECTIVE: This study was conducted to assess the characteristics of ESBL-producing Escherichia coli isolated from retail meats and shrimp in Nha Trang, Vietnam. MATERIALS AND METHODS: A total of 350 food samples (poultry [n=143], pork [n=147], and shrimp [n=60]) were purchased in July and November 2013 from a local market. ESBL-producing E. coli were isolated, and ESBL genotypes, phylogenetic groups, and antibiotic resistance profiles were determined. RESULTS: The prevalence of ESBL-producing E. coli in retail foods was 40.6%. ß-Lactamase-encoding genes of the CTX-M-1 (50.7%), CTX-M-9 (41.5%), TEM (59.9%), and SHV (2.8%) groups were detected singly or in combination. The percentages of single ESBL isolates harboring CTX-M-1 or -9 plus TEM groups were 35.2% and 16.2%, respectively. B1 was the most prevalent phylogroup in ESBL isolates from pork (44.7%), poultry (55.9%), and shrimp (72.7%). B2 was the least prevalent (4.2% and 4.8% for pork and poultry isolates, respectively). The prevalence of multidrug resistance (MDR; resistance to ≥ 3 antimicrobial groups) in ESBL-producing E. coli isolated from food was 85.9%. DISCUSSION AND CONCLUSIONS: This is the first report of the characteristics of ESBL-producing E. coli in retail foods in a local city in Vietnam. Our findings indicate that retail foods are contaminated with ESBL-producing E. coli, of which many were MDR. Further monitoring and public health efforts targeting food administration are needed to control the spread of ESBL-producing bacteria in communities.