Re-sensitization of mcr carrying multidrug resistant bacteria to colistin by silver.

Colistin is considered the last-line antimicrobial for the treatment of multidrug-resistant gram-negative bacterial infections. The emergence and spread of superbugs carrying the mobile colistin resistance gene (mcr) have become the most serious and urgent threat to healthcare. Here, we discover that silver (Ag+), including silver nanoparticles, could restore colistin efficacy against mcr-positive bacteria. We show that Ag+ inhibits the activity of the MCR-1 enzyme via substitution of Zn2+ in the active site. Unexpectedly, a tetra-silver center was found in the active-site pocket of MCR-1 as revealed by the X-ray structure of the Ag-bound MCR-1, resulting in the prevention of substrate binding. Moreover, Ag+effectively slows down the development of higher-level resistance and reduces mutation frequency. Importantly, the combined use of Ag+ at a low concentration with colistin could relieve dermonecrotic lesions and reduce the bacterial load of mice infected with mcr-1­carrying pathogens. This study depicts a mechanism of Ag+ inhibition of MCR enzymes and demonstrates the potentials of Ag+ as broad-spectrum inhibitors for the treatment of mcr-positive bacterial infection in combination with colistin.

Comparative analysis of outer membrane vesicles from uropathogenic Escherichia coli reveal the role of aromatic amino acids synthesis proteins in motility.

Uropathogenic Escherichia coli (UPEC) are causative agent that causes urinary tract infections (UTIs) and the recent emergence of multidrug resistance (MDR) of UPEC increases the burden on the community. Recent studies of bacterial outer membrane vesicles (OMV) identified various factors including proteins, nucleic acids, and small molecules which provided inter-cellular communication within the bacterial population. However, the components of UPEC-specific OMVs and their functional role remain unclear. Here, we systematically determined the proteomes of UPEC-OMVs and identified the specific components that provide functions to the recipient bacteria. Based on the functional network of OMVs' proteomes, a group of signaling peptides was found in all OMVs which provide communication among bacteria. Moreover, we demonstrated that treatment with UPEC-OMVs affected the motility and biofilm formation of the recipient bacteria, and further identified aromatic amino acid (AAA) biosynthesis proteins as the key factors to provide their movement.

Gold drugs as colistin adjuvants in the fight against MCR-1 producing bacteria.

The emergence and rapid spread of the mobile colistin resistance gene mcr-1 among bacterial species and hosts significantly challenge the efficacy of "last-line" antibiotic colistin. Previously, we reported silver nitrate and auranofin serve as colistin adjuvants for combating mcr-1-positive bacteria. Herein, we uncovered more gold-based drugs and nanoparticles, and found that they exhibited varying degree of synergisms with colistin on killing mcr-1-positive bacteria. However, pre-activation of the drugs by either glutathione or N-acetyl cysteine, thus releasing and accumulating gold ions, is perquisite for their abilities to substitute zinc cofactor from MCR-1 enzyme. X-ray crystallography and biophysical studies further supported the proposed mechanism. This study not only provides basis for combining gold-based drugs and colistin for combating mcr-1-positive bacterial infections, but also undoubtedly opens a new horizon for metabolism details of gold-based drugs in overcoming antimicrobial resistance.

Antimicrobial susceptibility of Bacteroides fragilis group organisms in Hong Kong, 2020-2021.

OBJECTIVES: This retrospective study analyzed the susceptibility levels of Bacteroides fragilis group (BFG) in a hospital-based laboratory where disk diffusion test (DDT) was routinely performed. Isolates non-susceptible to imipenem and metronidazole by DDT were further investigated using a gradient method. METHODS: The DDT and MIC susceptibility data of clindamycin, metronidazole, moxifloxacin and imipenem obtained on Brucella blood agar for 1264 non-duplicated isolates during 2020-2021 were analyzed. Species identification was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and 16S rRNA sequencing. Interpretative agreement of DDT results using the 2015 EUCAST tentative and 2021 CA-SFM breakpoints was compared against MIC as the reference. RESULTS: The dataset included 604 B. fragilis (483 division I, 121 division II isolates), 415 non-fragilis Bacteroides, 177 Phocaeicola and 68 Parabacteroides. Susceptibility rates for clindamycin (22.1-62.1%) and moxifloxacin (59.9-80.9%) were low and many had no inhibition zones. At the EUCAST and CA-SFM breakpoints, 83.0 and 89.4% were imipenem-susceptible, and 89.6% and 97.4 were metronidazole-susceptible. MIC testing confirmed 11.4% and 2.8% isolates as imipenem-non-susceptible and metronidazole-resistant, respectively. Significant numbers of false-susceptibility and/or false-resistance results were observed at the CA-SFM breakpoint but not the EUCAST breakpoint. Higher rates of imipenem and/or metronidazole resistance were detected in B. fragilis division II, B. caccae, B. ovatus, B. salyersiae, B. stercoris and Parabacteroides. Co-resistance to imipenem and metronidazole was detected in 3 B. fragilis division II isolates. CONCLUSIONS: The data demonstrated emerging BFG resistance to several important anti-anaerobic antibiotics and highlights the importance of anaerobic susceptibility testing in clinical laboratories to guide therapy.

Comprehensive investigation of antibiotic resistance gene content in cfiA-harboring Bacteroides fragilis isolates of human and animal origins by whole genome sequencing.

INTRODUCTION: The emergence of multidrug resistance in Bacteroides fragilis, especially the phylogenetic lineage carrying the carbapenemase gene cfiA, represents an increasing threat to human health. However, knowledge on the diversity of the multidrug-resistant strains and the genetic elements carrying the antibiotic resistance genes (ARGs) remains limited. AIM: The objective of the study was to describe the resistome in cfiA-positive B. fragilis. METHODS: A collection of cfiA-positive B. fragilis from diverse human (8 bacteremias, 15 wound infections) and animal (2 chickens, 2 pigs, 6 dogs, 3 cats) sources in Hong Kong, 2015-2017 was analysed by whole genome sequencing. RESULTS: In the 36 isolates, 13 distinct ARGs (total number 83, median 2, range 0-7 per isolate) other than cfiA were detected. ARGs encoding resistance to aminoglycosides, ß-lactams, macrolides, sulphonamides and tetracyclines were carried by CTn341-like, CTnHyb-like, Tn5220-like, Tn4555-like and Tn613-like transposons and were detected in phylogenetically diverse isolates of different host sources. Only few ARGs encoding resistance to metronidazole and tetracyclines were localized on plasmids. In two chicken isolates, a novel transposon (designated as Tn6994) was found to be involved in the dissemination of multiple ARGs mediating resistance to multiple antibiotics, including metronidazole and linezolid that are critically important for treatment of anaerobic infections. In mating experiments, Tn6994 and the associated phenotypic resistance could be transferred to Bacteroides nordii recipient. CONCLUSION: This study illustrates the importance of transposons in the dissemination of ARGs in the cfiA-positive division of B. fragilis. One Health approach is necessary to track the dissemination of ARGs.

Diversity of genomic clusters and CfiA/cfiA alleles in Bacteroides fragilis isolates from human and animals.

OBJECTIVES: To compare the phylogeny of cfiA-positive Bacteroides fragilis isolates from diverse human and animal sources. METHOD: Complete genome sequences were obtained from 42 cfiA-positive B. fragilis isolates (Hong Kong, 2015-2017) and additional 24 genomes deposited in the GenBank (multiple countries, 1985-2019) were included. The genomic clusters were constructed using PopPUNK. The CfiA alleles and polymorphism in the cfiA locus were analyzed in silico. RESULTS: The 66 isolates were grouped into 12 genomic clusters (BFSC-1 to 12). Human infection isolates were distributed in diverse clusters, being many of them common to fecal isolates from both human and animals. Thirteen CfiA alleles including 2 novel ones were identified. CfiA-1 (n = 28) is the predominating allele, following by CfiA-13 (n = 8), CfiA-4 (n = 7) and CfiA-14 (n = 6). The other CfiA alleles were identified in 1-3 isolates. Six patterns of gene context were identified in the regions flanking cfiA locus. No consistent association between genomic clusters and CfiA alleles could be detected. Similarly, markedly elevated imipenem MIC was linked to the integration, immediately upstram of cfiA of an IS element but not the CfiA allele or gene context. CONCLUSION: The phylogeny of cfiA-positive B. fragilis isolates causing human diseases was diverse and overlaped with those from human and animal carriage.

Rapid Broad Spectrum Detection of Carbapenemases with a Dual Fluorogenic-Colorimetric Probe.

Carbapenems stand as one of the last-resort antibiotics; however, their efficacy is threatened by the rising number and rapid spread of carbapenemases. Effective antimicrobial stewardship thus calls for rapid tests for these enzymes to aid appropriate prescription and infection control. Herein, we report the first effective pan-carbapenemase reporter CARBA-H with a broad scope covering all three Ambler classes. Using a chemical biology approach, we demonstrated that the absence of the 1ß-substituent in the carbapenem core is key to pan-carbapenemase recognition, which led to our rational design and probe development. CARBA-H provides a dual colorimetric-fluorogenic response upon carbapenemase-mediated hydrolysis. A clear visual readout can be obtained within 15 min when tested against a panel of carbapenemase-producing Enterobacteriaceae (CPE) clinical isolates that notably includes OXA-48 and OXA-181-producing strains. Furthermore, CARBA-H can be applied to the detection of carbapemenase activity in CPE-spiked urine samples.

Genomic investigation of a Streptococcus pneumoniae serotype 24F strain causing meningoencephalitis in Hong Kong.

Pneumococcal conjugate vaccines (PCVs) successfully decreased the incidence of invasive pneumococcal disease in children. However, many countries have reported serotype replacement and a rebound in diseases from non-vaccine serotypes. Here, we report the genomic investigation of a Streptococcus pneumoniae strain M215 that caused severe meningoencephalitis in an infant in 2019. The strain was assigned to serotype 24F using the bioinformatic pipeline SeroBA and pneumococcal type specific anti-sera. The strain was resistant to cotrimoxazole from mutations in both folA and folP genes. It was susceptible to penicillin and other non-ß-lactam antibiotics. Phylogenetically, it belongs to Global Pneumococcal Sequence Cluster (GPSC) 6 and multi-locus sequence type 162. A total of 38 virulence genes were detected in the genome of M215. Upon comparison of the profile of virulence genes, GPSC6 but not non-GPSC6 strains of serotype 24F and related serotypes were found to possess the major virulence determinant, pilus islet-1, comprising genes encoding sortases (srtB, srtC, srtD), pilus proteins (rrgA, rrgB and rrgC) and one transcriptional regulator (rlrA), which was previously described to be characteristic feature of international clones in the pre-PCV era. In our locality, this represented the first detection of serotype 24F and GPSC6/ST162 causing serious pneumococcal disease. The emergence of the non-vaccine serotype 24F GPSC6/ST162 lineage with molecular feature of high virulence is concerning and emphasizes the need for full characterization of strains causing severe disease.

The use of multi-pronged screening strategy to understand the epidemiology of carbapenemase-producing Enterobacteriaceae in Hong Kong: transition from epidemic to endemic setting.

A multi-pronged carbapenemase-producing Enterobacteriaceae (CPE) screening strategy was implemented in Hong Kong West healthcare network. Of 199,192 fecal specimens from 77,194 patients screening from 1 July 2011 to 30 June 2019, the incidence of CPE per 1000 patient admission significantly increased from 0.01 (2012) to 1.9 (2018) (p<0.01). With appropriate infection control measures, the incidence of nosocomial CPE per 1000 CPE colonization day decreased from 22.34 (2014) to 10.65 (2018) (p=0.0094). Exposure to wet market for purchasing raw pork (p=0.007), beef (p=0.017), chicken (p=0.026), and vegetable (p=0.034) for >3 times per week significantly associated with community acquisition of CPE. Strategic CPE control measures should be implemented in both the hospital and the community.

Modelling the impact of travel restrictions on COVID-19 cases in Hong Kong in early 2020.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) led to pandemic that affected almost all countries in the world. Many countries have implemented border restriction as a public health measure to limit local outbreak. However, there is inadequate scientific data to support such a practice, especially in the presence of an established local transmission of the disease. OBJECTIVE: To apply a metapopulation Susceptible-Exposed-Infectious-Recovered (SEIR) model with inspected migration to investigate the effect of border restriction as a public health measure to limit outbreak of coronavirus disease 2019. METHODS: We apply a modified metapopulation SEIR model with inspected migration with simulating population migration, and incorporating parameters such as efficiency of custom inspection in blocking infected travelers in the model. The population sizes were retrieved from government reports, while the number of COVID-19 patients were retrieved from Hong Kong Department of Health and China Centre for Disease Control (CDC) data. The R0 was obtained from previous clinical studies. RESULTS: Complete border closure can help to reduce the cumulative COVID-19 case number and mortality in Hong Kong by 13.99% and 13.98% respectively. To prevent full occupancy of isolation facilities in Hong Kong; effective public health measures to reduce local R0 to below 1.6 was necessary, apart from having complete border closure. CONCLUSIONS: Early complete travel restriction is effective in reducing cumulative cases and mortality. However, additional anti-COVID-19 measures to reduce local R0 to below 1.6 are necessary to prevent COVID-19 cases from overwhelming hospital isolation facilities.

Antibiotic prescriptions by medical interns in Hong Kong: influence of the hospital settings and prescription culture.

BACKGROUND: Inappropriate antibiotic prescribing practices predispose to resistance emergence. Despite the inclusion of the topic in medical school curricula worldwide, it is uncertain whether newly graduated medical interns have confidence in proper antibiotic prescription. OBJECTIVES: This study aimed to explore the antibiotic prescribing behaviours of the medical interns in Hong Kong and their barriers to appropriate antibiotic prescription. METHODS: Two focus groups were conducted among medical interns with training experiences in different public hospitals. Their prescribing behaviours and barriers were further examined with a questionnaire survey just before completion of internship. RESULTS: Focus group interviews identified a variety of hospital workplace cultures, including inappropriate empirical prescriptions and dosages, interns' passive roles in prescribing antibiotics and varied guidelines between different departments. Defensive medicine and lack of clinical experience were other barriers encountered. The interns believed that the incorrect practice learnt would perpetuate in their minds and affect their future practice. The top barriers reported by the survey respondents were adaptation to prescription culture of different hospitals (93.5%), lack of experience in antibiotic prescription (88.3%), inadequate knowledge in the choice of antibiotics (85.7%) and compliance with the seniors' instructions (80.6%). However, some focus group participants perceived weaker barriers in paediatric departments which provided close monitoring of antibiotic use. CONCLUSIONS: Inadequate knowledge and low confidence in antibiotic prescription led to the passive role of medical interns in antibiotic prescription, predisposing to future inappropriate practice. Inconsistent guidelines and prescription cultures between different hospitals and departments might further exacerbate their barriers.

Suppression of Staphylococcus aureus virulence by a small-molecule compound.

Emerging antibiotic resistance among bacterial pathogens has necessitated the development of alternative approaches to combat drug-resistance-associated infection. The abolition of Staphylococcus aureus virulence by targeting multiple-virulence gene products represents a promising strategy for exploration. A multiplex promoter reporter platform using gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus-virulence-associated genes was used to identify compounds that modulate the expression of virulence factors. One small-molecule compound, M21, was identified from a chemical library to reverse virulent S. aureus into its nonvirulent state. M21 is a noncompetitive inhibitor of ClpP and alters α-toxin expression in a ClpP-dependent manner. A mouse model of infection indicated that M21 could attenuate S. aureus virulence. This nonantibiotic compound has been shown to suppress the expression of multiple unrelated virulence factors in S. aureus, suggesting that targeting a master regulator of virulence is an effective way to control virulence. Our results illustrate the power of chemical genetics in the modulation of virulence gene expression in pathogenic bacteria.

A novel incompatibility group X3 plasmid carrying blaNDM-1 encodes a small RNA that regulates host fucose metabolism and biofilm formation.

The emergence of New Delhi metallo-beta-lactamase (NDM-1) has become a major health threat to clinical managements of gram-negative bacteria infections. A novel incompatibility group X3 plasmid (IncX3) pNDM-HN380 carrying blaNDM-1 has recently been found to epidemiologically link with multiple geographical areas in China. In this paper, we studied the metabolic responses of host bacteria E. coli J53 upon introduction of pNDM-HN380. A reduction of bacterial motility was observed in J53/pNDM-HN380. We profiled the RNA repertoires of the transconjugants and found a downregulation of genes involved in flagella and chemotaxis metabolic pathways at logarithmic (log) phase. We also identified a novel intragenic region (IGR) small RNA plas2. The plasmid-transcribed sRNA IGR plas2 was further characterized as a regulator of fucRwhich controls the fucose metabolism. By knockdown of IGR plas2 using an antisense decoy, we managed to inhibit the formation of bacterial biofilm of the host. Our study demonstrated a potential way of utilizing plasmid-transcribed sRNA against infectious bacteria.

Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of pncA PCR Sequencing.

An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtime MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

Genomic investigation of a sequence type 67 Clostridium difficile causing community-acquired fulminant colitis in Hong Kong.

In 2017, we identified a Clostridium difficile strain HKCD4 that caused community-acquired fulminant colitis in a previously healthy child. Phylogenetically, it belonged to clade 2, sequence type 67 and was resistant to fluoroquinolone and tetracycline. The strain was pathogenicity locus and binary toxin positive. It has a mutation in the trehalose repressor treR leading to the L172I substitution that was previously reported in the epidemic ribotype 027 lineage. HKCD4 has a tcdB sequence that shared very high identities with 3 highly virulent reference strains. It has a CpG depleted genome that is characteristic of hypervirulent C. difficile. The emergence of ST67 lineage with molecular feature of hypervirulence in the community is concerning and emphasizes the need for full characterization of strains causing severe disease in patients without classical risk factors.

Whole-genome sequencing data-based modeling for the investigation of an outbreak of community-associated methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit in Hong Kong.

We describe a nosocomial outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) ST59-SCCmec type V in a neonatal intensive care unit (NICU) in Hong Kong. In-depth epidemiological analysis was performed by whole-genome sequencing (WGS) of the CA-MRSA isolates collected from patients and environment during weekly surveillance and healthcare workers from the later phase of the outbreak. Case-control analysis was performed to analyze potential risk factors for the outbreak. The outbreak occurred from September 2017 to February 2018 involving 15 neonates and one healthcare worker. WGS analysis revealed complicated transmission dynamics between patients, healthcare worker, and environment, from an unrecognized source introduced into the NICU within 6 months before the outbreak. In addition to enforcement of directly observed hand hygiene, environmental disinfection, cohort nursing of colonized and infected patients, together with contact tracing for secondary patients, medical, nursing, and supporting staff were segregated where one team would care for CA-MRSA-confirmed/CA-MRSA-exposed patients and the other for newly admitted patients in the NICU only. Case-control analysis revealed use of cephalosporins [odds ratio 49.84 (3.10-801.46), p = 0.006] and length of hospitalization [odds ratio 1.02 (1.00-1.04), p = 0.013] as significant risk factors for nosocomial acquisition of CA-MRSA in NICU using multivariate analysis. WGS facilitates the understanding of transmission dynamics of an outbreak, providing insights for outbreak prevention.

A mixed-methods study on toilet hygiene practices among Chinese in Hong Kong.

BACKGROUND: Public toilets are a common transmission vector of infectious diseases due to environmental contamination. Research on Chinese people's hygiene practices in public lavatories are lacking. This study examined Chinese people's hygiene practices in public lavatories in Hong Kong. METHODS: We conducted qualitative interviews and a self-administered questionnaire survey with local residents from June 2016 to April 2018. Four focus group discussions and three individual interviews informed the design of the questionnaire. We recruited interviewees and survey respondents via social service centers. The interviews and questionnaire focused on the public's daily practices and hygiene behaviors in public toilets. Content analysis of qualitative data was conducted. Multivariable logistic regressions were used to examine the association between age and toilet hygiene behaviors. RESULTS: Our qualitative component revealed a range of handwashing practices, from not washing at all, washing without soap, to washing for a longer time than instructions. Other toilet use practices were identified, such as not covering toilet lid before flushing and stepping on toilet seats due to dirtiness, and spitting into toilet bowls or hand basin. Totally, 300 respondents completed the questionnaire. Among them, 212 (70.9%) were female and 246 (86.1%) were aged 65 or below. More than two thirds always washed hands with soap (68.7%) and dried hands with paper towels (68.4%). Up to 16.2% reported stepping on toilet seats and 43.9% never covered the toilet lid before flushing. Over one fourth (26.4%) spit into squat toilets/ toilet bowl. Regression analyses showed that the elderly group were less likely to report stepping on toilet seats (adjusted odds ratio, AOR = 0.17, 95%CI 0.03-0.88), flushing with the toilet lid closed (AOR = 0.40, 0.16-0.96), but more likely to spit into squat toilets/ toilet bowl (AOR = 4.20, 1.50-11.74). CONCLUSIONS: Hong Kong Chinese's compliance to hygiene practices in public toilets is suboptimal. Stepping on toilet seat is a unique Chinese practice due to the dirtiness of toilet seats. Spitting practices may increase the risk of airborne infectious diseases and need improvement. Measures are needed to improve toilet hygiene behaviors, including public education campaigns and keeping toilet environment clean.

IncX3 Epidemic Plasmid Carrying blaNDM-5 in Escherichia coli from Swine in Multiple Geographic Areas in China.

Six imported pigs originating from Guangdong, Henan, and Hunan provinces in China during October 2015 to February 2017 were cultured and found to be positive for meropenem-resistant Escherichia coli The samples yielded 9 E. coli isolates of diverse sequence types carrying blaNDM-5 on IncX3 (8 isolates from 5 farms) or IncFII (1 isolate from 1 farm) plasmids. The mcr-1 gene was coharbored by 4 isolates. The IncX3 plasmids (∼46 kb) carrying blaNDM-5 were identical or nearly identical to each other.

Rapid Detection of Carbapenemase Production in Enterobacteriaceae by Use of a Modified Paper Strip Carba NP Method.

Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) is important for preventing their spread in health care settings. We compared the performance of the Carba NP (CNP) test using the CLSI tube method with that using a modified paper strip method for the detection of carbapenemases in 390 Enterobacteriaceae isolates. The isolates were identified by Hong Kong's carbapenem-resistant Enterobacteriaceae surveillance program in 2016 and comprised 213 CPE and 177 carbapenemase-negative Enterobacteriaceae isolates. Molecular genotype was used as the reference. The test results were read at different time points for the CLSI method (1 min, 5 min, 1 h, and 2 h) and strip method (1 min and 5 min). The strip CNP and CLSI CNP tests correctly detect carbapenemase production in 93% and 93% of KPC producers, 100% and 38% of IMI producers, 94% and 85% of IMP producers, 98% and 90% of NDM producers, and 29% and 12% of OXA producers, respectively. Overall, the strip method has superior sensitivity to the CLSI method (86% versus 75%, respectively; P < 0.001, McNemar test). The specificity of both methods was 100%. By the CLSI method, 27%, 14%, 29%, and 6% of the CPE isolates were positive at 1 min, 5 min, 1 h, and 2 h, respectively. In contrast, by the strip method, 76% of the CPE isolates were positive at 1 min, and an additional 10% were positive at 5 min. In conclusion, the Carba NP test by use of the modified strip method has a higher sensitivity and a shorter assay time than that those by use of the CLSI tube method.

Global Distribution of Invasive Serotype 35D Streptococcus pneumoniae Isolates following Introduction of 13-Valent Pneumococcal Conjugate Vaccine.

A newly recognized pneumococcal serotype, 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG, which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background, and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of the wciG gene revealed 23 isolates from carriage (n = 4) and disease (n = 19) with partial or complete loss-of-function mutations, including mutations resulting in premature stop codons (n = 22) and an in-frame mutation (n = 1). These were selected for further analysis. The putative 35D isolates were geographically widespread, and 65.2% (15/23) of them was recovered after the introduction of pneumococcal conjugate vaccine 13 (PCV13). Compared with serotype 35B isolates, putative serotype 35D isolates have higher invasive disease potentials based on odds ratios (OR) (11.58; 95% confidence interval[CI], 1.42 to 94.19 versus 0.61; 95% CI, 0.40 to 0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% versus 7.6%; P = 0.009). Using the Quellung reaction, 50% (10/20) of viable isolates were identified as serotype 35D, 25% (5/20) as serotype 35B, and 25% (5/20) as a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype, 35D, among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule.