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Colistin is a last-line drug for multidrug-resistant Gram-negative bacteria. We previously reported four plasmid-mediated colistin resistance () gene-negative colistin-resistant clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-30Rx (isolates SRE34 and SRE44; MIC for colistin = 16 mg/liter), non-x (SME296; MIC = 8 mg/liter), and O18-ST416 (SME222; MIC = 4 mg/liter). In this study, we investigated the colistin resistance mechanism and identified novel amino acid substitutions or deletions in the PmrAB two-component system that activates (encoding a phosphoethanolamine transferase) and (encoding an undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase) in all colistin-resistant isolates. SRE34 possessed deletion Δ27-45 (LISVFWLWHESTEQIQLFE) in PmrB, SRE44 possessed substitution L105P in PmrA, and both SME222 and SME296 included substitution G206D in PmrB. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that lipid A is modified with phosphoethanolamine in all four isolates. Deletion of decreased colistin MICs to 0.5 mg/liter and lowered and expression. Chromosomal replacement of mutated or in colistin-susceptible O25b:H4-ST131 strain SME98 (colistin MIC = 0.5 mg/liter) increased the colistin MIC to that of the respective parent colistin-resistant isolate. In addition, SME98 mutants in which was replaced with mutated showed no significant differences in bacterial growth and competition culture from the parent strain, except for the mutant with L105P in PmrA, whose growth was significantly suppressed in the presence of the parent strain. In conclusion, some O25b:H4-ST131 strains appear to acquire colistin resistance via phosphoethanolamine modification of lipid A through amino acid changes in PmrAB, and the amino acid changes in PmrB do not influence bacterial growth.

The long-term clinical course, prognosis, and optimal management of symptoms and conditions after the acute phase of coronavirus disease 2019 remain to be elucidated. The purpose of this study was to clarify the characteristics of patients referred to a COVID-19 aftercare (CAC) clinic established at a tertiary academic hospital in Japan. MethodsThis study was a descriptive case series study. All patients who visited the CAC clinic between February 15 and September 17 in 2021 were included. Patients' background, chief complaints, and clinical courses after the onset of COVID-19 were described. ResultsA total of 87 Japanese patients (median age, 40.0 years; interquartile range [IQR], 26.5-53.0 years; 52.9% women) were referred to the CAC clinic. The median interval between the onset of COVID-19 and the visit to the clinic was 79.0 (IQR, 52.5-112.0) days. Referral sources were hospitals (36 patients), clinics (47 patients), a local healthcare center (3 patients), and other (1 patient). The most common chief complaint was general fatigue (50.4%) followed by dysosmia (28.7%), dysgeusia (26.4%), hair loss (18.4%), headache (17.2%), dyspnea (16.1%), and dyssomnia (13.1%). Respiratory symptoms were common in the early stages of the disease but were less common as the chief complaints when visiting the clinic. On the other hand, neurological, psychiatric, and extremity symptoms were predominant one month after the onset of COVID-19. ConclusionsRegardless of the severity in the acute phase, patients visiting our CAC clinic suffered from a variety of symptoms. General physicians skilled in using a comprehensive approach would be optimal to see patients with such complex symptoms.

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β-Lactam-resistant is a clinical concern. A high prevalence (>40%) of β-lactamase-negative high-level ampicillin-resistant (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of clinical isolates (62/104 isolates) were β-lactamase-negative ampicillin-resistant (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

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Aims:  Lactose intolerance, a serious health problem for Asians, can be solved using probiotic bacteria having high lactose hydrolysis activities. We determined the distribution of β‐galactosidase (β‐gal), phospho‐β‐galactosidase (P‐βgal) and phospho‐β‐glucosidase (P‐β‐glc) activities in species of lactic acid bacteria (LAB) isolated from human faeces to select strains for potential use in fermented dairy products, e.g. yogurt. Methods and Results:  The sugar substrates, o‐nitrophenyl‐β‐d‐ galactopyranoside 6‐phosphate and o‐nitrophenyl‐β‐d‐glucopyranoside 6‐phosphate, were synthesized and used to measure respectively P‐β‐gal and P‐β‐glc activities. Sixty‐five toluene‐treated strains were examined for three lactase enzyme activities. Lactobacillus mucosae OLL2848 showed the highest β‐gal activity (107·09 U mg−1 of protein) among the Lactobacillus strains from human faeces. Lactobacillus gasseri OLL2836 and OLL 2948 showed the highest P‐β‐gal (46·58 U) and P‐β‐glc (50·19 U)activity, respectively, with no β‐gal activity. Conclusions:  The expression of P‐β‐glc induced by lactose was characteristic of Lact. gasseri. Because this LAB is a major inhabitant of the human intestine. This enzyme is a key glycosidase involved in lactose utilization. Significance and Impact of Study:  This is the first report describing the distribution of three glycosidase activities used in lactose metabolism in LAB isolated from human faeces for possible use in functional foods.

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