A rabbit monoclonal antibody-mediated lateral flow immunoassay for rapid detection of CTX-M extended-spectrum ß-lactamase-producing Enterobacterales.

Infections of CTX-M extended-spectrum ß-lactamase-producing Enterobacterales are a severe threat in clinical settings. CTX-M genes on plasmids have been transferred to many Enterobacterales species, and these species have spread, leading to the global problem of antimicrobial resistance. Here, we developed a lateral flow immunoassay (LFIA) based on an anti-CTX-M rabbit monoclonal antibody. This antibody detected CTX-M variants from the CTX-M-9, CTX-M-2, and CTX-M-1 groups expressed in clinical isolates. The LFIA showed 100% sensitivity and specificity with clinical isolates on agar plates, and its limit of detection was 0.8 ng/mL recombinant CTX-M-14. The rabbit monoclonal antibody did not cross-react with bacteria producing other class A ß-lactamases, including SHV. In conclusion, we developed a highly sensitive and specific LFIA capable of detecting CTX-M enzyme production in Enterobacterales. We anticipate that our LFIA will become a point-of-care test enabling rapid detection of CTX-M in hospital and community settings as well as a rapid environmental test.

Prevalence and mechanism of fluoroquinolone resistance in clinical isolates of Proteus mirabilis in Japan.

Fluoroquinolone (FQ) and cephalosporin (CEP) resistance among Enterobacteriaceae has been increasingly reported. FQ resistance occurs primarily through mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). CEP resistance in Enterobacteriaceae is mainly due to the production of CTX-M type extended-spectrum ß-lactamases. Although prevalence and mechanisms of FQ and CEP resistance in Enterobacteriaceae such as Escherichia coli have been well studied, little is known about Proteus mirabilis in Japan. In this study, we assessed the prevalence and mechanism of FQ resistance in Japanese clinical isolates of P. mirabilis. We collected 5845 P. mirabilis isolates from eight hospitals between 2000 and 2013. Prevalence of FQ resistance was calculated as the annual average percentage of all P. mirabilis isolates. We selected 50 isolates exhibiting susceptibility, intermediate resistance, or resistance to levofloxacin (LVX) and identified amino acid substitutions in GyrA, GyrB, ParC, and ParE. The prevalence of FQ-resistant P. mirabilis gradually increased from 2001 to 2004, reaching 16.6% in 2005, and has remained relatively high (13.3-17.5%) since then. Low-level LVX-resistant strains (MIC, 8-16 mg/L) showed significant changes in GyrB (S464Y or -I, or E466D). High-level LVX-resistant strains (MIC, 32-128 mg/L) displayed significant changes in GyrA (E87K) and ParE (D420N). The highest-level LVX-resistant strains (MIC, ≥ 256 mg/L) presented significant changes in GyrA (E87K or -G), GyrB (S464I or -F), and ParE (D420N). Our findings suggest that substitutions in GyrA (E87) and ParE (D420) have played an important role in the emergence of high-level LVX-resistant P. mirabilis isolates (MIC, ≥ 32 mg/L) in Japan.

[Multidrug-Resistant Acinetobacter baumannii].

Teikyo University Hospital reported an outbreak of multidrug-resistant Acinetobacter baumannii (MDRAB) to the local public health department in 2010. The number of inpatients with MDRAB including asympto- matic carriers was 58 between August 2009 and September 2010. The way to tackle infection control issues has been comprehensively revised since this event in our hospital. The change could not have been achieved by a single department, such as the Department of Infection Control and Prevention or the Central Laboratory alone. Rather, collaboration among every department in the hospital was necessary. Although the impact of the outbreak on our hospital was enormous, it elucidated various clues to improve hospital man- agement regarding not only infection control but also safety management. [Review].

Genotyping of Acinetobacter baumannii strains isolated at a Japanese hospital over five years using targeted next-generation sequencing.

Acinetobacter baumannii is a Gram-negative bacterial agent involved in nosocomial infections. In this five-year retrospective study, phylogenetic relationships among carbapenem-resistant A. baumannii strains that were isolated at Teikyo University Hospital in Tokyo metropolis, Japan, were explored. A panel of 72 carbapenem-resistant A. baumannii strains that isolated from January 2006 until August 2010 was studied. Next-Generation sequencing (NGS) was employed to perform large-scale genotyping of these isolates. They were separated, according to the time of isolation, into two genetically distinct groups, one correspondent to strains of the outbreak reported to local public health department in 2010 and the other contained strains from earlier isolations, suggesting different origins of the isolates. Moreover, taxa in each group showed two main clustering patterns. Multilocus sequence typing (MLST) study on 8 isolates from the last outbreak showed that they were from one sequence type, 92, displaying less discriminatory power comparing to large-sequence typing. The clonal lineage profiles produced in this retrospective study will be used as a reference database to compare future isolations of A. baumannii. This study demonstrates the power of NGS in conducting epidemiological researches, allowing a high resolution genotyping.

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes.

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis-causing non-dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non-dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non-azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.

Recurrent Helicobacter cinaedi cellulitis and bacteremia in a patient with systemic lupus erythematosus.

A 31-year-old woman who had developed systemic lupus erythematosus at 17 years of age was admitted to the hospital for suspected cellulitis in the lower extremities. A blood culture performed upon admission to the hospital detected Helicobacter cinaedi (H. cinaedi), which was also isolated in blood and fecal cultures obtained on the 42nd hospital day. Bacterial translocation of H. cinaedi present in the intestines may have led to the development of recurrent bacteremia and cellulitis. In cases such as this, appropriate antibiotics therapy might be needed for more than one month. Moreover, H. cinaedi, a cause of emerging infections, requires a long period of time to grow; therefore it is important to extend the culture duration when the presence of this bacterium is suspected.

Rapid identification of Mycoplasma pulmonis isolated from laboratory mice and rats using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Mycoplasma species identification is based on biochemical, immunological, and molecular methods that require several days for accurate identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method for identification of bacteria and has recently been introduced into the clinical microbiology laboratory as a rapid and accurate technique. This method allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycoplasmal cells. In this study, we evaluated the performance of the MALDI-TOF MS for the identification of Mycoplasma by comparison with standard sequence analysis of 16S rRNA. We developed the first database of MALDI-TOF MS profiles of Mycoplasma species, containing Mycoplasma pulmonis, M. arthritidis, and M. neurolyticum, which are the most common pathogens in mice and/or rats, and species-specific spectra were recorded. Using the database, 6 clinical isolates were identified. Six tracheal swabs from 4 mice and 2 rats were cultured on PPLO agar for 4 to 7 days, and the colonies were directly applied to analyze the protein profiles. Five strains were identified as M. pulmonis, and 1 strain from a mouse was identified as M. neurolyticum (spectral scores were >2.00); the results were consistent with the results of the 16S rRNA gene sequence analysis (homologies>97.0%). These data indicate that MALDI-TOF MS can be used as a clearly rapid, accurate, and cost-effective method for the identification of M. pulmonis isolates, and this system may represent a serious alternative for clinical laboratories to identify Mycoplasma species.

The usefulness of changing focus during examination using Gram staining as initial diagnostic clue for infective tuberculosis.

Gram staining is a useful technique for detecting bacteria but is highly questionable in detecting Mycobacterium tuberculosis. Its detection generally requires special staining, such as Ziehl-Neelsen staining. We experienced three cases in which tuberculosis was first suggested by Gram staining of sputum or pus, confirmed by Ziehl-Neelsen staining, and diagnosed by polymerase chain reaction or culture. To find colorless tubercle bacilli in clinical samples with various organisms, varying the focus to slightly longer and shorter during study of the slides is indispensable. We present criteria for detecting infective pulmonary tuberculosis in Gram staining. First, in the ordinary focus, weakly stained, thin, gram-positive bacilli are found; second, with a slightly longer focus distance, the thin, cord-like, conspicuous gram-positive bacilli can be observed; and third, with a shorter focus distance, the gram-positive bacilli have changed into the brightened, colorless, or ghost ones. Four laboratory technologists each evaluated 20 Gram-stained samples after being lectured on the criteria, with no prior information about the sample. They accurately evaluated the presence of the bacilli in Gram-stained preparations in more than 90% of samples containing 3+ bacilli on Ziehl-Neelsen staining. Gram staining is available as an easy and rapid initial clue to recognize highly infective tuberculosis.