Inhibition of Bacterial Efflux Pumps by Crude Extracts and Essential Oil from Myristica fragrans Houtt. (Nutmeg) Seeds against Methicillin-Resistant Staphylococcus aureus.

Myristicafragrans Houtt. (Nutmeg) is a widely known folk medicine across several parts of Asia, particularly used in antimicrobial treatment. Bacterial resistance involves the expression of efflux pump systems (chromosomal norA and mepA) in methicillin-resistant Staphylococcus aureus (MRSA). Crude extract (CE) and essential oil (EO) obtained from nutmeg were applied as efflux pump inhibitors (EPIs), thereby enhancing the antimicrobial activity of the drugs they were used in. The major substances in CE and EO, which function as EPIs, in a descending order of % peak area include elemicin, myristicin, methoxyeugenol, myristicin, and asarone. Here, we investigated whether the low amount of CE and EO used as EPIs was sufficient to sensitize MRSA killing using the antibiotic ciprofloxacin, which acts as an efflux system. Interestingly, synergy between ciprofloxacin and CE or EO revealed the most significant viability of MRSA, depending on norA and mepA, the latter being responsible for EPI function of EO. Therefore, CE and EO obtained from nutmeg can act as EPIs in combination with substances that act as efflux systems, thereby ensuring that the MRSA strain is susceptible to antibiotic treatment.

Synergistic effect of vancomycin combined with cefotaxime, imipenem, or meropenem against Staphylococcus aureus with reduced susceptibility to vancomycin

Background/aim: We investigated the synergistic effect between vancomycin and ß-lactams against vancomycin-susceptible (VSSA) and nonsusceptible MRSA isolates [heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA]. Materials and methods: A total of 29 MRSA, including 6 VISA, 14 hVISA, and 9 VSSA isolates, were subjected to a microbroth dilu- tion-minimum inhibitory concentration (MIC) checkerboard using vancomycin combined with cefotaxime, imipenem, or meropenem. To confirm synergistic activity, the representative strains of VISA, hVISA, and VSSA were then selected for the time-kill curve method. Results: The combination of vancomycin with imipenem, meropenem, or cefotaxime exhibited synergistic effects against 17 (2 VISA, 9 hVISA, and 6 VSSA), 14 (3 VISA, 9 hVISA and 2 VSSA), and 5 (3 VISA and 2 hVISA) isolates, respectively. Additive and indifferent effects were found in the remaining isolates, but no antagonistic effect was observed. Using time-kill assay, the vancomycin combined with either imipenem or cefotaxime demonstrated synergism against both VISA and hVISA isolates, while the synergistic effect with meropenem was obtained only in the VISA isolates. Conclusion: This study demonstrated in vitro enhanced antibacterial activity of vancomycin plus ß-lactams against clinical hVISA or VISA isolates. These combinations may be an alternative treatment for MRSA infections in clinical practice.

Direct detection of methicillin-resistant in Staphylococcus spp. in positive blood culture by isothermal recombinase polymerase amplification combined with lateral flow dipstick assay.

Methicillin-resistant staphylococci (MRS) are important antimicrobial-resistant pathogens in sepsis. Conventional blood cultures take 24-72 h. The polymerase chain reaction (PCR)-based methods give faster results (2-3 h) but need expensive thermal cyclers. We therefore developed an isothermal recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay for rapid detection of MRS in spiked blood culture samples. Fifty-six clinical isolates including 38 mecA-carrying staphylococci and 18 non-mecA-carrying organisms as confirmed by PCR methods were studied. RPA primer set and probe specific for mecA gene (encoding penicillin-binding protein 2a) were designed. RPA reaction was carried out under isothermal condition (45 °C) within 20 min and read by LFD in 5 min. The RPA-LFD provided 92.1% (35/38) sensitivity for identifying MRS in positive blood culture samples, and no cross-amplification was found (100% specificity). This test failed to detect three mecA-carrying S.sciuri isolates. The detection limits of RPA-LFD method for identifying MRS were equal to those of PCR method. The RPA-LFD is simple, fast, and user-friendly. This method could detect the mecA gene directly from the positive blood culture samples without requirement for special equipment. This method would be useful for appropriate antibiotic therapy and infection control, particularly in a low-resource setting.

Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital.

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC50 and MIC90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 µg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC50 and MIC90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried blaOXA-23-like. The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

Attenuated total reflection: Fourier transform infrared spectroscopy for detection of heterogeneous vancomycin-intermediate Staphylococcus aureus.

Staphylococcus aureus strains resistant to the last line antibiotic, vancomycin, have been of clinical concern. These include heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA. The hVISA phenotype cannot be detected by routine laboratory methods. Characterization of hVISA/VISA by new technologies is necessary to differentiate them rapidly from the vancomycin-susceptible isolates (VSSA). In this study, we developed a model for discrimination of hVISA from VSSA by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with multivariate data analysis, displaying a phenotypic signature of the bacteria. ATR-FTIR spectra were acquired from a total of 59 clinical methicillin-resistant S. aureus (MRSA) isolates comprising 28 hVISA and 31 VSSA strains. Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were used to analyze 351 spectra of 39 isolates and develop a discrimination model for identifying hVISA and VSSA. The classification model, which was used for blind testing of 90 spectra from each of 10 hVISA, and 10 VSSA isolates, provided 100% sensitivity and specificity. The modeling revealed that the major discrimination between hVISA and VSSA phenotypes involved bands related to cell wall content (1087 and 1057 cm-1). This study showed that ATR-FTIR technique may be an alternative method for rapid detection of low-level vancomycin-resistant S. aureus.

A novel GoldNano Carb test for rapid phenotypic detection of carbapenemases, particularly OXA type, in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp.

Objectives: To develop a simple gold nanoparticle (AuNP)-based colorimetric test, GoldNano Carb (GoldC), for detecting carbapenemase production in Gram-negative bacteria, compared with updated Carba NP (CNP) and CarbAcineto NP (CAcNP) tests by using PCR methods as gold standard. Methods: Ninety-nine carbapenemase-producing Enterobacteriaceae (CPE), Pseudomonas spp. and Acinetobacter spp. isolates and 89 non-CPE isolates were tested by the GoldC and CNP. Additionally, the CAcNP was performed in the Acinetobacter spp. isolates. The final imipenem (imipenem/cilastatin form) concentration was 5 mg/mL for all three tests. For the GoldC, the imipenem powder was added directly to bacterial cell suspension in distilled water prior to detection of acid product by the citrate-capped AuNP solution. An AuNP change from red to purple, blue or green indicates carbapenemase activity. Results: The GoldC detected all carbapenemase producers except one OXA-23-like producer (99.0% sensitivity), whereas 11 carbapenemase producers (10 Acinetobacter and 1 P. aeruginosa) were CNP negative (88.9% sensitivity). However, the GoldC and CNP provided 100% and 98.6% sensitivity, respectively, for the CPE and Pseudomonas spp. Both tests gave one false positive from CTX-M-1-like-producing Enterobacter spp. (98.9% specificity). The GoldC and CAcNP detected 96.7% and 93.3% of the Acinetobacter spp. isolates, respectively. Interestingly, times to positivity by the GoldC were markedly shorter than those by the CNP (76.8% versus 36.2% positive at 5 min) and CAcNP (43.3% at 5 min versus 20% within 30 min). Conclusions: The GoldC is fast, easy, highly sensitive and inexpensive (∼$0.25 per test), suggesting that it may be suitable for routine carbapenemase detection in low-resource settings for infection control or epidemiological purposes.

Rapid and simple identification of carbapenemase genes, bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups, in Gram-negative bacilli by in-house loop-mediated isothermal amplification with hydroxynaphthol blue dye.

Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla NDM-1, 9 bla IMP-14a, 2 bla IMP-48, 1 bla IMP-1, 1 bla IMP-4, 1 bla IMP-9, 1 bla IMP-15, 4 bla VIM-2, 1 bla VIM-1, 1 bla IMP-14a & bla VIM-2, 7 bla KPC-2, 3 bla OXA-48 and 3 bla OXA-181) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.

Modification and evaluation of the Carba NP test by use of paper strip for simple and rapid detection of carbapenemase-producing Enterobacteriaceae.

Carbapenemase-producing Enterobacteriaceae (CPE) isolates have now emerged worldwide. We therefore modified the phenotypic Carba NP test by use of a filter paper strip for easily and rapidly identifying CPE in routine laboratory. A collection of 56 CPE and carbapenemase-producing Pseudomonas spp. isolates (including 28 NDM-1, 11 IMP-14a, 1 IMP-1, 1 IMP-4, 1 IMP-9, 1 IMP-15, 4 VIM-2, 1 VIM-1, 1 IMP-14a with VIM-2, 3 OXA-48, 3 OXA-181 and 1 KPC-2 producers) and 41 non-CPE isolates (including 19 ESBL, 7 pAmpC, 3 AmpC, 9 ESBL with pAmpC and 3 non-ESBL & non-AmpC producers) as confirmed by the PCR methods were tested by the paper strip method using pharmaceutical imipenem/cilastatin as a substrate. Bacterial colonies of each isolate were applied directly on filter paper strips dropped with either imipenem-phenol red (test strip) or phenol red solution alone (control strip). The reaction was read within 5 min. This test failed to detect 3 OXA-181, 2 OXA-48 and 3 IMP-14a producers (85.7 % sensitivity), whereas no false positives were seen (100 % specificity). Further evaluation of the paper strip test in 267 CPE screening-positive isolates from three hospitals by their medical technologists showed 92.0 % sensitivity (100 % for NDM producers) and 100 % specificity compared with the PCR methods. Because of its ease, rapidness and cost effective, the paper strip test has a potential for routine CPE testing in low-resource laboratories particularly in areas with high prevalence of NDM enzymes, leading to appropriate antimicrobial therapy and infection control strategy.

Rapid detection of Staphylococcus aureus in blood culture samples using human IgG-based lateral flow assay.

Staphylococcus aureus is one of the most common pathogens. The conventional workflow for identifying this organism is time-consuming and takes up to several days. Therefore, we developed a colloidal gold-based lateral flow immunoassay (LFIA) using human IgG as a conjugated antibody to detect S. aureus. One hundred and thirty-eight clinical isolates, including 79 S. aureus and 59 non-S. aureus were spiked in blood samples, and incubated at 37°C for 24 h. The bacterial antigens were simply extracted before being tested by the developed LFIA strips. The results were read by the naked eye within 15 min. Conventional PCR was used as a reference method. The sensitivity and specificity of the developed LFIA were 100% (95% CI: 94.2%-100.0% and 92.4%-100.0%, respectively) in spiked blood culture samples. The detection limits of the LFIA for the purified protein A and bacterial colonies were 10-3 µg/mL and 107 CFU/mL, respectively. The performance of the LFIA testing in 221 bacterial colony isolates and 118 positive blood culture bottles from three hospitals by their medical technologists showed 98.1% (95% CI: 94.1%-99.5%) and 89.7% (95% CI: 79.3%-95.4%) sensitivity, respectively. The LFIA is a quick, easy, and sensitive method for detecting S. aureus without expensive equipment. It might have the potential for early diagnosis of routine service in low-resource laboratories, leading to a rapid and effective treatment.IMPORTANCEIn this study, we modified our previously developed lateral flow immunoassay (LFIA) test for the detection of Staphylococcus aureus by using an in-house human IgG as a conjugated antibody instead of the specific commercial antibody. It gave comparable results to the former developed-LFIA test and helped cost reduction.

ST9 MRSA strains carrying a variant of type IX SCCmec identified in the Thai community.

BACKGROUND: Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in Thailand occur most frequently in healthcare facilities. However, reports of community-associated MRSA are limited. METHODS: We characterized 14 MRSA isolates from outpatients (O-1 to O-14) by phenotypic and genotypic methods and compared them with 5 isolates from inpatients (I-1 to I-5). Thai MRSA isolates from a healthcare worker (N-1) and a pig (P-1) were also included as ST9 MRSA strains from other sources. RESULTS: All MRSA isolates from the outpatients and inpatients were multidrug-resistant (resistant to ≥3 classes of antimicrobials). All of them except strains O-2 and I-3 carried type III SCCmec and belonged to agrI, coagulase IV, spa type t037 or t233, which related to ST239. The strain O-2 (JCSC6690) carried type IX SCCmec and belonged to agrII, coagulaseXIc, spa type t337 and ST9, whereas the strain I-3 carried a type III SCCmec and belonged to ST1429. Nucleotide sequence determination revealed that the type IX SCCmec element in strain O-2 was distinct from that in a Thai ST398 strain (JCSC6943) previously identified in 2011; nucleotide identities of ccrA and ccrB were 93 and 91%, respectively and several open reading frames (ORFs) at the joining regions were different. PCR experiments suggested that strain O-2 and N-1 carried similar SCCmec element, whereas that of strain P-1 was different, suggesting that distinct ST9-MRSA-IX clones might be spreading in this province. CONCLUSIONS: The SCCmecIX-ST9 MRSA clones of distinct SCCmec subtypes might have emerged in the Thai community and might also have disseminated into the hospital.

Emergence of NDM-1- and IMP-14a-producing Enterobacteriaceae in Thailand.

OBJECTIVES: To detect carbapenemases in clinical isolates of Enterobacteriaceae collected from patients in a university hospital in Thailand between October 2010 and August 2011. METHODS: A total of 4818 Enterobacteriaceae isolates were screened for the presence of carbapenemases by ertapenem and imipenem disc diffusion tests. All positive screening isolates were subjected to modified Hodge test, phenylboronic acid- and EDTA-carbapenem combined disc tests and two multiplex PCRs of bla(IMP), bla(VIM), bla(SPM), bla(SIM) and bla(GIM), and of bla(KPC), bla(NDM) and bla(OXA-48). Carbapenemase-producing isolates were typed by PFGE and then characterized by antimicrobial susceptibility tests. Conjugation was performed using a broth culture mating method. RESULTS: Two isolates each of Escherichia coli, Klebsiella pneumoniae and Citrobacter freundii produced NDM-1, whereas two other isolates of K. pneumoniae produced IMP-14a. DNA fingerprints revealed that the metallo-ß-lactamase (MBL)-producing isolates were of different strains except for clonal strains of C. freundii. In vitro transfer of carbapenem resistance was successful for the eight MBL-producing isolates. All MBL producers were susceptible to colistin and tigecycline. The six NDM-producing isolates were recovered from the urine of three patients, who had no history of travel outside Thailand. Interestingly, one patient had chronic urinary tract infections caused by a K. pneumoniae strain and two strains of E. coli producing NDM-1. CONCLUSIONS: Surveillance of carbapenemases, particularly NDM-1, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in our country.

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.

Effect of Vancomycin on Cellular Fatty Acid Profiles of Vancomycin-Susceptible and Nonsusceptible Staphylococcus aureus.

Vancomycin is widely used for treatment of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) leading to an increasing appearance of low-level vancomycin-resistant isolates called heterogeneous vancomycin-intermediate S. aureus (hVISA). The mechanism of vancomycin tolerance in hVISA is still unclear. This study aimed to investigate the fatty acid compositions of S. aureus isolates under the stress environment with vancomycin. The different responses of hVISA and vancomycin-susceptible S. aureus (VSSA) may lead to more understanding the mechanism. The bacterial lipid profiles were tested three times from three extractions of each isolate cultured on tryptic soy agar (TSA) and TSA with vancomycin. Of the 30 MRSA isolates studied, 13, 12, and 5 isolates were VSSA, hVISA, and VISA, respectively. The analysis of bacterial lipid profiles showed that under vancomycin stress, there was a reduction of straight chain fatty acids (SCFAs) in VSSA isolates but an increase in branched chain fatty acids (BCFAs). In contrast, the hVISA group exhibited an increase only in the BCFAs but not in SCFAs. Of interest, vancomycin had no effect on either BCFAs or SCFAs of the VISA cells. This study provided information of bacterial adaptation during stress with vancomycin that may be helpful to overcome the resistant bacteria.

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Rapid Detection of OXA-48-like Carbapenemase Genes in Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) possessing various carbapenemases, particularly the OXA-48 group, are now rapidly spreading and becoming a major public health concern worldwide. Phenotypic detection of OXA-48-like carbapenemases is still suboptimal due to their weak carbapenemase activity, whereas highly sensitive and specific polymerase chain reaction (PCR)-based methods take at least 3-4 h. We, therefore, developed a recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip assay for the rapid detection of blaOXA-48-like in Enterobacterales. A total of 131 clinical isolates including 61 blaOXA-48-like-carrying Enterobacterales isolates and 70 Gram-negative bacilli isolates containing other bla genes were subjected to the RPA method performed under isothermal conditions at 37 °C within 10 min and visually inspected by LF strip within 5 min. The RPA-LF assay provided 100% sensitivity (95% confidence interval, 92.6-100%) and 100% specificity (93.5-100%) for detecting blaOXA-48-like genes from bacterial colonies. Its detection limit was 100 times less than that of the PCR method. This assay is rapid, easy to perform, and provides excellent performance without any special equipment. It may be applied for directly identifying the blaOXA-48-like genes in Enterobacterales obtained from blood culture. Rapid identification of carbapenemase types is essential for selecting appropriate antimicrobial options, particularly the ß-lactams combined with novel ß-lactamase inhibitors.

Multi Evaluation of a Modified GoldNano Carb Test for Carbapenemase Detection in Clinical Isolates of Gram-Negative Bacilli.

Carbapenemase-producing Gram-negative bacteria have been increasingly reported. Simple and sensitive methods for carbapenemase detection are still needed. In this study, a gold nanoparticle (AuNP) solution was modified by the addition of zinc sulfate (ZnSO4) for improving the conventional GoldNano Carb (cGoldC) test, and the modified GoldC (mGoldC) test was then evaluated for phenotypic detection of carbapenemase production in Gram-negative bacilli clinical isolates. ZnSO4 was added to give final concentrations of 0.25, 0.5, 0.75, and 1 mM. The performance of the mGoldC test was evaluated in Enterobacterales, Acinetobacter spp., and Pseudomonas aeruginosa isolates from six hospitals in different regions using polymerase chain reaction (PCR) as a gold standard. The AuNP solution with 0.25 mM ZnSO4 was used for the mGoldC test. Evaluation of the mGoldC test in 495 Enterobacterales, 212 Acinetobacter spp., and 125 P. aeruginosa isolates (including 444 carbapenemase producers and 388 non-carbapenemase producers) revealed sensitivity, specificity, a positive likelihood ratio, and a negative likelihood ratio of 98.6%, 98.2%, 54.7, and 0.01, respectively. This test is fast, easy to perform, cost-effective (~0.25 USD per test), and highly sensitive and specific for routine carbapenemase detection, thus leading to effective antimicrobial therapy and infection control measures.

Colistin Susceptibility Testing by Rapid Colistin Disk Elution Test Among Enterobacteriaceae in Low-Resource Setting.

We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-µg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 µg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-µL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

Rapid detection of Enterococcus and vancomycin resistance using recombinase polymerase amplification.

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

Preliminary report of SCCmec-types and antimicrobial susceptibilities of methicillin-resistant Staphylococcus aureus isolates from a university hospital in Thailand.

Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide. It is a major cause of hospital-acquired infections in most hospitals for nearly half century. The present study was conducted to examine the antimicrobial susceptibilities and staphylococcal cassette chromosome mec (SCCmec)-type for MRSA isolates from 237 patients treated at Srinagarind Hospital between September 2002 and August 2003. Antimicrobial susceptibility testing for all isolates was performed using an agar dilution method and SCCmec-types of 81 representatives from 237 isolates were determined using multiplex PCR. The minimum inhibitory concentration (MIC) ranges for the MRSA isolates were as follows: cefazolin 8 to > or =64; erythromycin < or = 0.5 to > or =64; gentamicin < or = 0.5 to > or =64; imipenem < or = 0.5 to >16; ofloxacin < or = 0.5 to > or =64; oxacillin 16 to > or =64; tetracycline 2 to > or =64 and vancomycin < or = 0.5 to 2 microg/ml. All MRSA isolates were susceptible to vancomycin, but only 0.4% to 8.9% was susceptible to the remaining antimicrobial agents. Of the 81 isolates tested, 2 types of SCCmec were found (76 with type III and 2 with type II) and no mecA gene was detected in 3 isolates. Sixty-seven of the 78 isolates carried the mercury-resistant operon. The multilocus sequence type in isolates with type III SCCmec was ST239 and in isolates with type II SCCmec was ST5.

Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein A in Positive Blood Culture Samples.

Bloodstream infection (BSI) is a major cause of mortality in hospitalized patients worldwide. Staphylococcus aureus is one of the most common pathogens found in BSI. The conventional workflow is time consuming. Therefore, we developed a lateral flow immunoassay (LFIA) for rapid detection of S. aureus-protein A in positive blood culture samples. A total of 90 clinical isolates including 58 S. aureus and 32 non-S. aureus were spiked in simulated blood samples. The antigens were extracted by a simple boiling method and diluted before being tested using the developed LFIA strips. The results were readable by naked eye within 15 min. The sensitivity of the developed LFIA was 87.9% (51/58) and the specificity was 93.8% (30/32). When bacterial colonies were used in the test, the LFIA provided higher sensitivity and specificity (94.8% and 100%, respectively). The detection limit of the LFIA was 107 CFU/mL. Initial evaluation of the LFIA in 20 positive blood culture bottles from hospitals showed 95% agreement with the routine methods. The LFIA is a rapid, simple and highly sensitive method. No sophisticated equipment is required. It has potential for routine detection particularly in low resource settings, contributing an early diagnosis that facilitates effective treatment and reduces disease progression.

PmrB mutations including a novel 10-amino acid repeat sequence insertion associated with low-level colistin resistance in carbapenem-resistant Acinetobacter baumannii.

The global emergence of colistin resistance in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates is a serious public health concern. We therefore aimed to investigate colistin resistance mechanisms in 5 colistin-resistant (COL-R) CRAB isolates collected from Thai patients in 2016 by whole genome sequencing (WGS) compared with those of 5 colistin-intermediate (COL-I) CRAB isolates from the same period. All isolates were subjected to antimicrobial susceptibility testing, efflux pump inhibitor-based test and WGS. Mutations in known genes associated with colistin resistance were analyzed and deleterious mutations were then predicted by PROVEAN tool. The 10 CRAB isolates carried blaOXA-23 with the addition of blaOXA-58 in 1 isolate. All COL-R isolates exhibited colistin MICs of 4 µg/mL except for 1 isolate with that of 16 µg/mL. They belonged to ST2, ST16, ST23, ST164 and ST215, whereas the COL-I isolates with colistin MICs of ≤0.25-1 µg/mL were ST2, ST164 and ST215. Neither increased efflux pump activity nor mcr gene was found in any COL-R isolate. Three COL-R isolates contained different PmrB variants: a novel 10-amino acid (aa) repeat sequence insertion, VILGCILIFS between positions 27 and 28 (S27_A28insVILGCILIFS) in transmembrane domain (TM); a 1-aa insertion, alanine between positions 162 and 163 (A162_I163insA) in TM; and a 1-aa substitution, A226T in histidine kinase domain. One COL-R isolate possessed PmrA variant with A80V substitution. These alterations were predicted as deleterious. Mechanisms of colistin resistance in the remaining COL-R isolate were still unknown. In conclusion, the alterations in both PmrB and PmrA were predicted and suggested as initial mutations responsible for low-level colistin resistance in our CRAB isolates. Under selective pressure, these isolates may exhibit higher level colistin resistance by the additional mutations, leading to more therapeutic difficulties.