A 12-year epidemiological study of Acinetobacter baumannii from blood culture isolates in a single tertiary-care hospital using polymerase chain reaction (PCR)-based open reading frame typing.

Objective: Acinetobacter baumannii is a causative agent of healthcare-associated infections, and the introduction and spread of A. baumannii that has acquired drug resistance within a hospital are serious healthcare problems. We investigated the transition of epidemic clones and the occurrence of outbreaks by molecular epidemiological analysis to understand the long-term behavior of A. baumannii within a single facility. Methods: A. baumannii isolates collected from blood-culture-positive patients between January 2009 and December 2020 were subjected to PCR-based open reading frame typing (POT) for species identification, clonal typing, and homology searches. Results: Of the strains isolated from blood cultures, 49 were identified as A. baumannii and analyzed with POT. The POT#1=122 clones had different antimicrobial resistance profiles to the other POT clones, and strains belonging to this clone were dominant during outbreaks of multidrug-resistant Acinetobacter. Although the clonal diversity of A. baumannii decreased and its antimicrobial resistance increased during the outbreaks, clonal diversity and the in-hospital antibiogram improved at the end of the outbreaks. The POT#1=122 clone was not eliminated from the hospital during the study period. Conclusions: POT is a simple and suitable method for molecular epidemiological monitoring and can show the introduction, outbreak, and subsequent transition of an epidemic clone of A. baumannii.

Erratum: A 12-year epidemiological study of Acinetobacter baumannii from blood culture isolates in a single tertiary-care hospital using polymerase chain reaction (PCR)-based open reading frame typing - ERRATUM.

[This corrects the article DOI: 10.1017/ash.2022.279.].

Single nucleotide polymorphisms in genes encoding penicillin-binding proteins in ß-lactamase-negative ampicillin-resistant Haemophilus influenzae in Japan.

OBJECTIVE: ß-Lactamase-negative ampicillin-resistant Haemophilus influenzae is a common opportunistic pathogen of hospital- and community-acquired infections, harboring multiple single nucleotide polymorphisms in the ftsI gene, which codes for penicillin-binding protein-3. The objectives of this study were to perform comprehensive genetic analyses of whole regions of the penicillin-binding proteins in H. influenzae and to identify additional single nucleotide polymorphisms related to antibiotic resistance, especially to ampicillin and other cephalosporins. RESULTS: In this genome analysis of the ftsI gene in 27 strains of H. influenzae, 10 of 23 (43.5%) specimens of group III genotype ß-lactamase-negative ampicillin-resistant H. influenzae were paradoxically classified as ampicillin-sensitive phenotypes. Unfortunately, we could not identify any novel mutations that were significantly associated with ampicillin minimum inhibitory concentrations in other regions of the penicillin-binding proteins, and we reconfirmed that susceptibility to ß-lactam antibiotics was mainly defined by previously reported SNPs in the ftsI gene. We should also consider detailed changes in expression that lead to antibiotic resistance in the future because the acquisition of resistance to antimicrobials can be predicted by the expression levels of a small number of genes.

Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsI mutation of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae.

Rapid and easy detection of sequence polymorphisms, including nucleotide point mutations of bacterial pathogens responsible for amino acid substitutions linked to drug resistance, is essential for the proper use of antimicrobial agents. Here, a detection method using loop-mediated amplification (LAMP) combined with amplification refractory mutation system (ARMS) to accurately distinguish a different single nucleotide in the target sequence was established, named ARMS-SNP LAMP. This procedure is capable of species-specific detection of a nucleotide (1578T) in the ftsI gene on Haemophilus influenzae without amplifying the sequence carrying the point mutations (T1578G/A) in ß-lactamase-negative ampicillin resistant (BLNAR) strains. Reactions were performed at 61°C for 45min. Successful target gene amplifications were detected by measuring real-time turbidity using a turbidimeter and visual detection. The assay had a detection limit of 10.0pg of genomic DNA per reaction and showed specificity against 52 types of pathogens, whereas amplifications were completely blocked in even 100.0ng/µL of genomic DNA with point mutations at T1578G and T1578A. The expected ARMS-SNP LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This novel procedure was also used to analyze 57 clinical isolates of H. influenzae. All 25 clinical isolates with the naïve sequence of 1578T gave positive results. In addition, concordant negative results were obtained for 31 of the BLNAR strains with the T1578G mutation and one strain with the T1578A mutation. The ARMS-SNP LAMP method is a simple and rapid method for SNP-genotyping of a clinical isolate as point-of-care testing (POCT) technology. It is suitable for use in both resource-limited situations and well-equipped clinical settings because of its simplicity and convenience.

Diagnosis of imported Ugandan typhoid fever based on local outbreak information: A case report.

Re-emerging multidrug-resistant typhoid fever is becoming a worldwide threat, especially in East Africa. At the beginning of 2015, an outbreak of typhoid fever started in the capital city of Uganda, and 1940 suspected cases were reported by 5 March 2015. In this report, we describe a case of typhoid fever caused by a MDR strain with HIV infection and hemoglobin S-syndrome thalassemia in an Ugandan from Kampala City. It is essential to consider MDR strains of Salmonella enterica serovar Typhi infections, including fluoroquinolone-resistant strains, in patients from Africa and Southeast Asia.

Evaluation and validity of a polymerase chain reaction-based open reading frame typing method to dissect the molecular epidemiology for Acinetobacter baumannii in an epidemiologic study of a hospital outbreak.

Acinetobacter baumannii is regarded as one of the most important pathogens in hospital outbreaks. To obtain an efficient and simple epidemiologic method of surveillance during outbreaks, we assessed the applicability of the polymerase chain reaction-based open reading frames typing (POT) method and compared it with pulsed-field gel electrophoresis. The POT method was found to have sufficient discriminatory power to identify the strains and would be widely applicable to epidemiologic surveillance during hospital outbreaks.

Blood stream infections caused by Acinetobacter baumannii group in Japan - Epidemiological and clinical investigation.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex, especially A. baumannii, Acinetobacter pittii and Acinetobacter nosocomialis, constitutes an important group of nosocomial pathogens; however, epidemiological or clinical characteristics and prognosis is limited in Japan. From 2009 to 2013, 47 blood stream infection cases resulting from A. baumannii group were reviewed at the National Defense Medical College, an 800-bed tertiary hospital. To determine the genospecies, further comparative nucleotide sequence analyses of the RNA polymerase b-subunit (rpoB) gene were performed. Sequence analysis of rpoB gene showed that 25 (49.0%), 17 (33.3%) and 5 (9.8%) cases were caused by A. baumannii, A. pittii and A. nosocomialis, respectively. The 30-day and in-hospital mortality rates of A. baumannii were 8.5% and 25.5%, respectively, and there were no significant differences between Acinetobacter species. Clinical characteristics were statistically insignificant. Multidrug-resistant Acinetobacter species were detected in 3 cases (5.9%) with same pulsed-field gel electrophoresis (PFGE) pattern and A. baumannii was less susceptible to amikacin and levofloxacin. In this study, the mortality and clinical characteristics were similar among A. baumannii group isolate cases despite some showing drug resistance. However, identification of Acinetobacter species helps to initiate appropriate antibiotic therapy in earlier treatment phase, because A. baumannii shows some drug resistance.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification.

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 °C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 10(3) cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

[Trend of susceptibility testing of clinical isolates of Candida species from aseptic samples in hospitals in Saitama prefecture].

We investigated the susceptibility of Candida species from clinical aseptic samples, including blood, at some hospitals in Saitama prefecture. Candida spp. detected from aseptic samples in the 6 institutes in Saitama prefecture from November 2007 to July 2011 were studied. The number of isolates was 85, which are 43 (50.6%) of Candida albicans, 24 (28.2%) of Candida parapsilosis, 5 (5.9%) of Candida glabrata, 5 (5.9%) of Candida tropicalis, 4 (4.7%) of Candida guilliermondii, 2 (2.4%) of Candida fermentati, 1 (1.2%) of Candida famata and Candida lusitaniae, respectively. All isolates were susceptible to amphotericin B. However, resistant isolates against micafungin were 3 in 5 of C. glabrata. We analyzed susceptibility of Candida spp. in Saitama prefecture in the article, and our study might be useful for the fungal therapy in the region.

Bacteremia due to Mycobacterium massiliense in a patient with chronic myelogenous leukemia: case report.

Bacteremia due to Mycobacterium massiliense is rare. We present here the case of a 58-year-old man with chronic myelogenous leukemia complicated by bacteremia caused by M. massiliense. The microorganisms were identified as M. massiliense by sequencing analysis, having initially been misdiagnosed as M. abscessus by DNA-DNA hybridization.