Emergence and Evolution of Unique Plasmids Harboring blaIMP-70 and blaCTX-M-253 in Multidrug-Resistant Providencia rettgeri.

Although the prevalence of carbapenem-resistant Enterobacterales remains low in Japan, these bacteria are a growing problem worldwide, owing to their multidrug resistance phenotype. We isolated a multidrug-resistant Providencia rettgeri strain, NR1418, harboring a rare blaIMP variant, blaIMP-70, a novel blaCTX-M variant, designated blaCTX-M-253, and blaMOX-1. This strain is resistant to ß-lactams, amikacin, levofloxacin, and colistin. Genomic analysis revealed that NR1418 carries two plasmids, designated pNR1418-1 and pNR1418-2. The pNR1418-1 plasmid harbors blaCTX-M-253, blaTEM-1, and blaMOX-1, while the pNR1418-2 plasmid harbors blaIMP-70, which is located in a class 1 integron. Both plasmids exhibit high similarities with the plasmid of the P. rettgeri isolate BML2526, which also harbors blaIMP-70 and was identified in the same region of Japan as NR1418 at a different point in time. This indicates the possibility of the emergence and evolution of IMP-70-producing P. rettgeri and suggests that the plasmid of BML2526 may have occurred following recombination of the two plasmids harbored by NR1418. Further, blaIMP-70 and blaCTX-M-253 were found on unique plasmids, indicating that they likely evolved through mutations and recombination. IMPORTANCE Although Providencia rettgeri is an opportunistic pathogen, its intrinsic resistance to colistin and tigecycline makes the treatment of carbapenem-resistant P. rettgeri challenging. We isolated a multidrug-resistant P. rettgeri strain which harbored a rare blaIMP variant, blaIMP-70, a novel blaCTX-M variant, blaCTX-M-253, and blaMOX-1 from a urinary sample obtained in Osaka, Japan. We investigated its genetic structure and evaluated the evolution of the plasmids carrying these genes. We show that blaIMP-70, blaCTX-M-253, and blaMOX-1 are present on unique plasmids and that they have high similarities to the plasmid of another IMP-70-producing P. rettgeri isolate that was identified as being from the same location. The evolution of plasmids through mutations and recombination may play a role in the development and spread of multidrug resistance.

Evaluation of NG-Test CARBA 5 for the detection of carbapenemase-producing Gram-negative bacilli.

Carbapenemase-producing Enterobacterales (CPE) pose one of the most serious antimicrobial resistance threats to public health worldwide. The outcome of CPE infection differs depending on the resistance mechanism. Therefore, rapid detection of CPE infection is essential for optimizing patient management. The carbapenem inactivation method (CIM) and modified CIM (mCIM) are standard methods for detecting CPE, but they usually require 24 h to generate results. Recently, an immunochromatographic assay, NG-Test CARBA 5, has become commercially available. It detects the five most common carbapenemase producers (KPC, IMP, NDM, VIM, and OXA-48) rapidly and accurately. We aimed to evaluate the diagnostic accuracy of NG-Test CARBA 5 for detecting carbapenemase-producing Gram-negative bacilli (CPGNB). We used 116 carbapenemase-producing strains and 48 non-carbapenemase-producing strains. Of the 116 carbapenemase-producing strains, 107 harboured genes for at least one of the five most common carbapenemases, KPC, IMP, NDM, VIM, and OXA-48-like. Forty-eight non-carbapenemase-producing strains, including carbapenem-resistant Enterobacterales, harboured genes for extended-spectrum ß-lactamases (CTX-M groups [n=25] and SHV groups [n=2]) or plasmid-mediated AmpC ß-lactamases (DHA [n=3], CMY-2 [n=2], and CFE-1 [n=1]). Antimicrobial susceptibility was tested using the agar dilution method, according to the Clinical and Laboratory Standards Institute guidelines. Of the 116 carbapenemase-producing strains, 79 were resistant to at least meropenem or imipenem. The sensitivity and specificity of the NG-Test CARBA 5 for the strains were 99.1 % (106 strains positive for 107 strains of the five most common carbapenemase producers) and 100 % (60 strains negative for other types of CPGNB [n=10] and non-CPGNB strains [n=48]), respectively. The carbapenemase-producing strain with a false-negative result produced IMP-66. The NG-Test CARBA 5 had high sensitivity and specificity for detecting carbapenemase-producing strains.

A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii.

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase ß-subunit gene sequence analysis. RESULTS: PCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain. CONCLUSIONS: This assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.

Development of a loop-mediated isothermal amplification assay for rapid Helicobacter pylori detection.

Infection with cagA-positive Helicobacter pylori is associated with gastric cancer. Molecular techniques are vital for accurate H. pylori diagnosis. We developed a loop-mediated isothermal amplification (LAMP) for detecting the H. pylori cagA gene and evaluated its use for clinical diagnosis. A LAMP primer set was designed to recognize the homologous regions of cagA gene sequences of 6 H. pylori strains. LAMP sensitivity was evaluated with serial dilutions of H. pylori ATCC 43504 and fecal specimens; specificity was evaluated with H. pylori ATCC 49396 and CIP 104086. The LAMP sensitivity for H. pylori specimens was 10-1 cfu/tube (reaction time, 37 min), which was 10-fold more sensitive than polymerase chain reaction. LAMP was also highly sensitive and rapid for fecal specimens. It detected cagA gene from ATCC 49396 and CIP 104086. The findings suggest LAMP can be used for diagnosing and screening of H. pylori infections to decrease gastric cancer incidence.

Molecular Epidemiology of Ampicillin-resistant Haemophilus influenzae Causing Acute Otitis Media in Japanese Infants and Young Children.

BACKGROUND: Nontypeable Haemophilus influenzae is a particularly important cause of acute otitis media (AOM). There is a high prevalence of ß-lactamase-nonproducing ampicillin-resistant (BLNAR) strains in Japanese children, which is associated with recurrent AOM and prolonged treatment. The aim of this study was to investigate the antimicrobial susceptibility profile, mechanisms of ampicillin resistance and molecular epidemiology of ampicillin resistance in H. influenzae strains causing AOM in Japanese children. METHODS: One hundred fifty-seven strains of H. influenzae isolated from the middle ear fluid of pediatric patients (aged 0-3 years) with AOM from various areas of Japan were studied. The antimicrobial susceptibility profile, genes encoding ß-lactamase and alterations of penicillin-binding protein 3 were investigated. Genetic relatedness among ampicillin-resistant isolates was examined by multilocus sequence typing and pulsed-field gel electrophoresis. RESULTS: Of 157 isolates, 108 (68.8%) demonstrated reduced susceptibility to ampicillin, including 95 (60.5%) of ß-lactamase-nonproducing isolates and 13 (8.3%) of ß-lactamase-producing isolates. All BLNAR (minimum inhibitory concentration of ampicillin ≥ 4 mg/L) isolates had amino acid substitutions related to ampicillin resistance. Multilocus sequence typing and pulsed-field gel electrophoresis demonstrated genetic diversity although there were 2 clusters of highly resistant isolates with identical STs (sequence types; ST161 and 549). CONCLUSIONS: Alterations of penicillin-binding protein 3 represented the most prevalent mechanism of ampicillin resistance among H. influenzae isolates causing AOM in Japanese children. BLNAR isolates from children with AOM demonstrated genetic diversity. This study identified for the first time ST clones associated with BLNAR H. influenzae causing AOM in Japanese children.

Effect of carryover of clot activators on coagulation tests during phlebotomy.

We investigated the effect of clot activators carried over from the serum tube on major coagulation tests during phlebotomy. First, blood specimens from 30 normal subjects were mixed with small amounts of fluid containing clot activators, and their effects on various coagulation tests were determined. Only the value of fibrin monomer complex displayed a remarkable change when thrombin-containing fluid was added to the blood specimens. Subsequently, 100 paired blood specimens (taken from 75 healthy volunteers and 25 patients taking warfarin) were collected in coagulation tubes before and after the serum tube using standard phlebotomy procedures. Various coagulation tests were performed to determine the effect of contamination of thrombin-containing blood on coagulation parameters. Differences between the 2 tubes were minimal but significant for some of the coagulation tests. Therefore, we conclude that the effect of clot activators in the serum tube on coagulation tests is minimal when standard phlebotomy procedures are used.