Kumiko Kawamura

Targeted Disruption of Gb3/CD77 Synthase Gene Resulted in the Complete Deletion of Globo-series Glycosphingolipids and Loss of Sensitivity to Verotoxins

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 μg of VT-2 or 1.0 μg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.

First Detection of Fosfomycin Resistance Gene fosA3 in CTX-M-Producing Escherichia coli Isolates from Healthy Individuals in Japan

We examined the prevalence and mechanism of fosfomycin resistance in CTX-M-producing Escherichia coli isolates from healthy Japanese individuals. One hundred thirty-eight CTX-M-producing E. coli isolates were subjected to fosfomycin susceptibility testing. The presence of acquired fosfomycin resistance genes such as fosA, fosA3, and fosC2 was explored, and the transmissibility of fosfomycin resistance, replicon type of plasmid, and genetic environment of fosA3 were investigated. Eight isolates (5.8%) showed resistance to fosfomycin, five of which harbored fosA3, which was in genetic linkage with blaCTX-M. The replicon types of the five transferred fosA3-carrying plasmids were as follows: IncI1 (n=2), IncN (n=1), and IncFII (n=2). Each fosA3 gene was located close to the blaCTX-M gene and was flanked by IS26 elements. These genetic environments of fosA3 in E. coli from healthy individuals were quite similar to those observed in the clinical and veterinary settings. Our results indicate that fosA3 genes possibly inserted by small mobile genetic elements flanked by two IS26 elements have already spread throughout the plasmids along with the blaCTX-M genes of commensal E. coli colonizing in healthy Japanese people.

Practical Agar-Based Disk Potentiation Test for Detection of Fosfomycin-Nonsusceptible Escherichia coli Clinical Isolates Producing Glutathione S-Transferases

ABSTRACT The number of reports concerning Escherichia coli clinical isolates that produce glutathione S-transferases responsible for fosfomycin resistance (FR-GSTs) has been increasing. We have developed a disk-based potentiation test in which FR-GST producers expand the growth inhibition zone around a Kirby-Bauer disk containing fosfomycin in combination with sodium phosphonoformate (PPF). PPF, an analog of fosfomycin, is a transition-state inhibitor of FosAPA, a type of FR-GST from Pseudomonas aeruginosa. Considering its mechanism of action, PPF was expected to inhibit a variety of FR-GSTs. In the presence of PPF, zone enlargement around the disk containing fosfomycin was observed for FosA3-, FosA4-, and FosC2-producing E. coli clinical isolates. Moreover, the growth inhibition zone was remarkably enlarged when the Mueller-Hinton (MH) agar plate contained 25 μg/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, ≥256 μg/ml) E. coli clinical isolates from our hospital with the potentiation test, 6 FR-GST producers were positive phenotypically by potentiation disk and were positive for FR-GST genes: 5 harbored fosA3 and 1 harbored fosA4. To identify the production of FR-GSTs, we set the provisional cutoff value, 5-mm enlargement, by adding PPF to a fosfomycin disk on the MH agar plates containing G6P. Our disk-based potentiation test reliably identifies FR-GST producers and can be performed easily; therefore, it will be advantageous in epidemiological surveys and infection control of fosfomycin-resistant bacteria in clinical settings.

Long-term Colonization of blaCTX-M-Harboring Escherichia coli Found in Ordinary Japanese People Engaged in Food Handling in Community

ABSTRACT The actual state of intestinal long-term colonization by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in healthy Japanese people remains unclear. Therefore, a total of 4,314 fecal samples were collected from 2,563 food handlers from January 2010 to December 2011. Approximately 0.1 g of each fecal sample was inoculated onto a MacConkey agar plate containing cefotaxime (1 μg/ml). The bacterial colonies that grew on each plate were checked for ESBL production by the double-disk synergy test, as recommended by the Clinical and Laboratory Standards Institute. The bacterial serotype, antimicrobial susceptibility, pulsotype, sequence type (ST), and ESBL genotype were checked, and the replicon types of plasmids harboring the ESBL gene were also determined after conjugation experiments. ESBL producers were recovered from 70 (3.1%) of 2,230 participants who were checked only once. On the other hand, ESBL producers were isolated at least once from 52 (15.6%) of 333 participants who were checked more than twice, and 13 of the 52 participants carried ESBL producers for from more than 3 months to up to 2 years. Fluoroquinolone (FQ)-resistant E. coli strains harboring bla CTX-M were repeatedly recovered from 11 of the 13 carriers of bla CTX-M-harboring E. coli. A genetically related FQ-resistant E. coli O25b:H4-ST131 isolate harboring bla CTX-M-27 was recovered from 4 of the 13 carriers for more than 6 months. Three FQ-resistant E. coli O1:H6-ST648 isolates that harbored bla CTX-M-15 or bla CTX-M-14 were recovered from 3 carriers. Moreover, multiple CTX-M-14- or CTX-M-15-producing E. coli isolates with different serotypes were recovered from 2 respective carriers. These findings predict a provable further spread of ESBL producers in both community and clinical settings.

Contribution of QnrA, a Plasmid-Mediated Quinolone Resistance Peptide, to Survival of Escherichia coli Exposed to a Lethal Ciprofloxacin Concentration

We evaluated the effects of qnrA on survival of bacteria exposed to a lethal ciprofloxacin (CIP) concentration and development of quinolone resistance through the accumulation of amino acid substitutions in quinolone resistance-determining regions (QRDRs) of GyrA and ParC, targets of quinolones, in Escherichia coli. CIP-susceptible E. coli strains of different O-serotypes (O1, O6, O18, O25b, O74, and O78) were transformed by a recombinant plasmid harboring qnrA, and the parent strains and their transformants were subjected to killing curve assays and adaptation tests. In the killing curve assay at 2 × the minimum inhibitory concentration of CIP, the viable bacterial cell numbers of strains O1, O6, and O25b were maintained at 10(5)-10(8) CFU/mL after 24-h incubation, while the remaining strains showed a 10(5)-fold reduction in viable cell numbers. In the adaptation test, a Ser83-Leu substitution in the QRDR of GyrA was identified earlier in the parent strains of O25b and O1 than in their transformants, suggesting that the acquisition of qnrA did not necessarily accelerate the rate of accumulation of amino acid substitutions in the QRDR. We confirmed that the presence of qnrA contributed to increased survival of the E. coli strains displaying certain O-serotypes. Further studies are necessary to evaluate the precise effects of qnrA on quinolone resistance acquisition by Enterobacteriaceae.

First detection of Escherichia coli harboring mcr-1 gene from retail domestic chicken meat in Japan

Global spread of the plasmid-mediated colistin resistance gene, mcr-1 poses a challenge to public health because colistin is the last-line-of-defense against severe infections of multidrug-resistant Gram-negative bacteria. In Japan, a few studies have reported the prevalence of mcr-1 among food animal-derived Escherichia coli isolates, but the prevalence of mcr-1 in retail meats is not well known. We report here the first detection of mcr-1 in retail chicken meat. A total of 70 extended-spectrum beta-lactamase-producing E. coli isolates, recovered from retail chicken meats between August 2015 and June 2016, were screened for mcr-1. We found 1 CTX-M-1 beta-lactamase-producing E. coli isolate belonging to ST1684, phylogroup A. The mcr-1 gene was not located on an IncI1 plasmid encoding the blaCTX-M-1 gene. However, whole plasmid sequencing revealed that mcr-1 was located on an IncI2 plasmid. The sequences of the nikB-mcr-1-pap2-ydfA-topB region of the IncI2 plasmid in this study was almost identical to that of the previously described IncI2 plasmid, pECJS-61-63 present in E. coli isolated from pig feces in China, except for containing a synonymous mutation in the mcr-1 gene. Plasmid carrying the mcr-1 gene have not yet been identified in human isolates in Japan. Thus, strict monitoring or surveillance of colistin resistance among Gram-negative bacteria recovered from retail meat of food animals under colistin pressure, and humans, is crucial.

Specific blaCTX-M-8/IncI1 Plasmid Transfer among Genetically Diverse Escherichia coli Isolates between Humans and Chickens

ABSTRACT We investigated the genetic backbones of 14 blaCTX-M-8-positive Escherichia coli isolates recovered from human stool samples and chicken meat. All isolates carried IncI1 plasmids with blaCTX-M-8 (blaCTX-M-8/IncI1), and most (9/14) belonged to a specific genetic lineage, namely, plasmid sequence type 113 (pST113). The genetic contexts of the nine blaCTX-M-8/IncI1 pST113 plasmids were similar, regardless of the source. These results suggest the probable local transfer of blaCTX-M-8/IncI1 between humans and chickens with genetically diverse E. coli.

Development of a novel chromogenic method, Penta-well test, for rapid prediction of β-lactamase classes produced in clinical Enterobacteriaceae isolates

We developed a novel chromogenic method, Penta-well test, which enables the rapid detection and classification of β-lactamases in clinical Enterobacteriaceae isolates. This test is based on a combination of nitrocefin and 3 β-lactamase inhibitors specific to classes A, B, and/or C, with nitrocefin hydrolysis by β-lactamases being assessed by optical density measurements at 490 nm. When the cutoff value for each β-lactamase class was determined (0.09, 0.4, and 0.55 for class A, class B, and class C β-lactamase producers, respectively), the sensitivity and specificity of classification were 93.5% and 68.8% for class A, 93.8% and 100% for class B, and 86.7% and 100% for class C, respectively. Moreover, this method allowed accurate β-lactamase classification in 20 of 23 (87.0%) isolates producing plural class of β-lactamases. Thus, the Penta-well test can provide information that would be useful in the accurate detection and classification of β-lactamases produced by causative bacteria.

Prevalence of CTX-M-Type Extended-Spectrum β-Lactamase-Producing Escherichia coli B2-O25-ST131 H30R Among Residents in Nonacute Care Facilities in Japan.

We investigated the prevalence and characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli among 258 residents of long-term care facilities (LTCFs) in Japan. Out of 258 fecal samples collected from nine LTCFs between November 2015 and March 2017, we recovered 59 ESBL-producing E. coli isolates. All isolates carried blaCTX-M genes, mainly blaCTX-M-27 (42.4%), blaCTX-M-14 (23.7%), and blaCTX-M-15 (18.6%). The isolates showed 7 serotypes (STs), including ST131 (n = 49, 83.1%) and ST38 (n = 4, 6.8%), and 47 (79.7%) out of 49 isolates belonging to ST131 were identified as H30R. The 59 ESBL producers were divided into four groups, B2 (86.4%), D (8.5%), A (3.4%), and C (1.7%); 44 (74.6%) were epidemic clone B2-O25-ST131 H30R, of which 21, 11, and 6 harbored blaCTX-M-27, blaCTX-M-15, and blaCTX-M-14, respectively. Most plasmids were of IncF replicon types (n = 33), and 22 blaCTX-M-27-carrying plasmids showed multiple replicon types, including IncFII, FIA, and FIB. The ESBL producers were susceptible to imipenem, amikacin, and fosfomycin, but resistant to ceftazidime (49.2%), and ciprofloxacin (88.1%); in particular, the isolates harboring the blaCTX-M-15 gene showed significantly high resistance rate to ceftazidime (p < 0.01). Our findings indicate that a considerable proportion of the examined LTCF residents carried ESBL-producing E. coli isolates in feces and had high prevalence of epidemic clone B2-O25-ST131. Furthermore, continuous investigations would be very necessary to monitor actual carriage states of ESBL-producers among the LTCF residents from the viewpoint of both public health and healthcare viewpoints.

High prevalence of blaCTX-M-14 among genetically diverse Escherichia coli recovered from retail raw chicken meat portions in Japan.

Global widespread of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, especially Escherichia coli poses a greater threat in healthcare and community settings of humans. Raw meats from food animals colonized with ESBL producers may be one of important transmission routes for those bacteria in the community. This study investigated the presence of ESBL-producing E. coli in retail raw chicken and pork meats in Japan. ESBL producers were detected from the 59 of 150 (39.3%) chicken samples, but none were from all the 50 pork samples tested. The blaCTX-M-14 (17; 24.3%) was most frequently identified, followed by blaCTX-M-2 (16; 22.9%), blaSHV-12 (11; 15.7%), and blaCTX-M-55 (10; 14.3%) among a total of 70 ESBL-producing E. coli isolates from 59 chicken samples. The isolates with blaCTX-M-14 were often combined with phylogroup B1 (9/17) mainly composed of ST162 (7/9), and phylogroup F (5/17) with diverse STs. The blaCTX-M-14 was basically associated with the common elements ISEcp1 and ΔIS903 or IS903 in all 17 isolates. In 6 isolates, comprising 5 phylogroup B1-ST162 and a nontypeable-ST162 isolates, an IS26-truncated ISEcp1 was identified upstream of the blaCTX-M-14, and a fosA3 was further located downstream of ΔIS903. Furthermore, some mobile genetic elements mediating blaCTX-M-14 unique to raw chicken meat portions were identified. The blaCTX-M-2 gene was preceded by ISEcp1 or ISCR1 in 16 isolates, whereas the presence of Δorf3 downstream of blaCTX-M-2 was limited only in 6 isolates from Brazilian samples though they exhibited diverse phylogroups and STs. The blaCTX-M-55 and blaCTX-M-1 shared classical flanking structures, ISEcp1-blaCTX-M-orf477, although the length of spacer sequences between ISEcp1 and the start codon of blaCTX-M was 45 bp and 80 bp for blaCTX-M-55 and blaCTX-M-1, respectively. Among blaSHV-12-harboring isolates, ST38 was frequently detected (6/11) though their phylogroup distribution varied. In conclusion, besides transmission of bla gene-harboring E. coli lineages which have adaptability to both human and chicken, spread of mobile genetic elements associated with bla genes from E. coli lineages adapted to chicken to those adapted to human is highly suggested. Our results provide important information to gain a better understanding of the transmission risk of bla genes from retail chicken meats to human.