Antibiotic-Resistance Genes in Waste Water.

Quinolone antibiotics represent one of the most important classes of anti-infective agents and, although still clinically valuable, their use has been compromised by the increasing emergence of resistant strains, which has become a prevalent clinical problem Quinolones act by inhibiting the activity of DNA gyrase and topoisomerase IV – two essential bacterial enzymes that modulate the chromosomal supercoiling required for critical nucleic acid processes The acquisition of quinolone resistance is recognized to be multifactorial and complex The main resistance mechanism consists of one or a combination of target-site gene mutations that alter the drug-binding affinity of target enzymes However, other mechanisms such as mutations that lead to reduced intracellular drug concentrations, by either decreased uptake or increased efflux, and plasmid-encoded resistance genes producing either target protection proteins, drug-modifying enzymes or multidrug efflux pumps are known to contribute additively to quinolone resistance The understanding of these different resistance mechanisms has improved significantly in recent years; however, many details remain to be clarified and the contribution of less-studied mechanisms still needs to be better elucidated in order to fully understand this phenotype

Mechanisms of quinolone action and resistance: where do we stand?

Clinically relevant evolution studies are needed to help fight the spread of antibiotic resistance For most of human history, bacterial pathogens have been a major cause of disease and mortality. The development of antibiotics provided a simple and effective treatment for bacterial infections, and antibiotics have since had huge effects on human health and longevity. These are threatened with the rise of antibiotic resistance (ABR): Many pathogenic bacteria have evolved resistance to the main classes of antibiotics, and multidrug-resistant bacteria have caused untreatable infections. ABR already imposes substantial health and economic burdens, and the global annual cost of ABR could increase to 10 million deaths and US$100 trillion by 2050 (1). Understanding how ABR evolves and spreads is therefore key to improving antibiotic treatment strategies.

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The evolution of antibiotic resistance

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory

Shigella spp. are a common cause of diarrheal disease and have remained an important pathogen responsible for increased rates of morbidity and mortality caused by dysentery each year around the globe. Antibiotic treatment of Shigella infections plays an essential role in reducing prevalence and death rates of the disease. However, treatment of these infections remains a challenge, due to the global rise in broad-spectrum resistance to many antibiotics. Drug resistance in Shigella spp. can result from many mechanisms, such as decrease in cellular permeability, extrusion of drugs by active efflux pumps, and overexpression of drug-modifying and -inactivating enzymes or target modification by mutation. Therefore, there is an increasing need for identification and evolution of alternative therapeutic strategies presenting innovative avenues against Shigella infections, as well as paying further attention to this infection. The current review focuses on various antibiotic-resistance mechanisms of Shigella spp. with a particular emphasis on epidemiology and new mechanisms of resistance and their acquisition, and also discusses the status of novel strategies for treatment of Shigella infection and vaccine candidates currently under evaluation in preclinical or clinical phases.

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Shigella: Antibiotic-Resistance Mechanisms And New Horizons For Treatment.

Bacteria can survive fluoroquinolone antibiotics (FQs) treatment by becoming resistant through a genetic change—mutation or gene acquisition. The SOS response is widespread among bacteria and exhibits considerable variation in its composition and regulation, which is repressed by LexA protein and derepressed by RecA protein. Here, we take a comprehensive review of the SOS gene network and its regulation on the fluoroquinolone resistance. As a unique survival mechanism, SOS may be an important factor influencing the outcome of antibiotic therapy in vivo .

SOS response and its regulation on the fluoroquinolone resistance.

The continuous emergence of carbapenem-resistant Escherichia coli (CRECO) presents a great challenge to public health. New Delhi metallo-lactamase (NDM) variants are widely disseminated in China, so the research on the prevalence and transmission of diverse blaNDM variants is urgently needed. In the present study, 54 CRECO isolates were collected from 1185 Escherichia coli isolates in five hospitals in Northern Jiangsu Province, China from September 2015 to August 2016. Antimicrobial susceptibility tests, PCR detection of resistance determinants, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to characterize these strains. Plasmid conjugation experiments were carried out to determine the transferability of resistant genes from selected isolates. PCR-based replicon typing (PBRT), S1 nuclease-PFGE and Southern blotting were conducted for plasmid profiling. Carbapenemase genes were detectable in all CRECO isolates, among which thirty-one CRECO isolates were found to carry blaNDM-5 (54.7%), while, blaNDM-1, blaNDM-7, blaNDM-4, blaNDM-9, and blaKPC-2 were identified in fourteen, five, two, one, and one isolate, respectively. MLST results revealed 15 different STs and four new STs were first reported to be linked with NDM-producing isolates. PFGE typing showed that no more than two isolates with the same ST appeared to the same band pattern except three ST410 isolates. Twenty-six selected NDM-producing isolates were successfully transferred to E. coli J53 by conjugation experiments. Notably, 50.0% (13/26) of blaNDM variants were found to be carried by ~55 kb IncX3 plasmid. Our study reported a high prevalence of blaNDM variants, especially blaNDM-5, in Northern Jiangsu province, China. Diverse blaNDM variants were mainly carried by ~55 kb IncX3 plasmids, suggesting that the fast evolution and high transferability of this kind of plasmid promote the high prevalence of blaNDM variants. Therefore, large-scale surveillance and effective infection control measures are also urgently needed to prevent diverse blaNDM variants from becoming epidemic in the future.

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High Prevalence of blaNDM Variants Among Carbapenem-Resistant Escherichia coli in Northern Jiangsu Province, China.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed as a new type of soft ionization mass spectrometry in recent years. An increasing number of clinical microbiological laboratories consider it as an innovative approach for bacterial identification. This study was undertaken in order to evaluate the use of MALDI-TOF MS for rapid identification of the clinical streptococci. A systematic review was conducted based on a literature search of the Medline and Embase databases. Fixed-effects models based on the P-value and the I-square were used for meta-analysis while considering the possibility of heterogeneity between studies. Statistical analyses were performed by using STATA 11.0. Twenty-seven studies covering 3,540 streptococci were included in our meta-analysis. The MALDI-TOF MS correctly identified the species of 96% (I2 = 92.8, P 0.1), and 100% of Streptococcus agalactiae (I2 = 20.7%, P > 0.2). What’s more, it also had high confidence in other Streptococcus. But the accuracy of bovis needs to be improved. The overall performance of both MALDI-MS systems was different. Notably, the identifying accuracy rate of streptococci by VITEK MS was 98%, compared to 94% by the MALDI biotyper system. Interestingly, when analyzing the incorrect identification of MALDI-TOF MS, 36 out of the 38 strains of Streptococcus mitis/oralis were inaccurately identified as Streptococcus pneumoniae by the MALDI biotyper system. In conclusion, the results of this review indicated that MALDI-TOF MS could be a reliable and rapid method for identification of the streptococci.

Performance of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry system for rapid identification of streptococci: a review.

The aim of this study was to evaluate the prevalence of fluoroquinolone resistance and mechanisms of selected fluoroquinolone resistance in Shigella flexneri isolates. A total of 624 S. flexneri strains isolated between 2001 and 2011 in Jiangsu Province of China were analysed for their fluoroquinolone susceptibility. The quinolone resistance-determining region of gyrA, gyrB, parC and parE were amplified and sequenced. In general, 90.5 % of S. flexneri exhibited resistance to nalidixic acid. The mean norfloxacin resistance rate was 22.4 % during the 11 years from 2001 to 2011 (6.4 % from 2001 to 2005 and 36.8 % from 2006 to 2011). Sequencing of gyrA, gyrB, parC and parE genes of all S. flexneri isolates showed that the mutation rate was as high as 93.9 %. In addition, 91.8 % and 92.3 % of S. flexneri harboured mutations in gyrA and parC, respectively. About 35.2 % of S. flexneri isolates susceptible to nalidixic acid contained mutations. Meanwhile, mutations were detected in 91.2 % of norfloxacin-susceptible strains, and almost all S. flexneri isolates resistant to fluoroquinolone contained mutations. To the best of our knowledge, this is the first study reporting the occurrence of point mutations Asn57Lys and His80Pro in gyrA and Ala85Thr, Asp111His and Ser129Pro in parC. Emerging fluoroquinolone resistance with a significantly high mutation rate of the gyrA and parC genes in S. flexneri in Jiangsu Province deserves attention, and monitoring antibiotic susceptibility is important for the effective management of S. flexneri infections.

Novel mutations in quinolone resistance-determining regions of gyrA, gyrB, parC and parE in Shigella flexneri clinical isolates from eastern Chinese populations between 2001 and 2011.

Purpose Carbapenem-resistant Klebsiella pneumoniae (CRKP) have emerged worldwide and also being a major threat to children and neonate. In this study, we describe a nosocomial outbreak of NDM-5-producing Klebsiella pneumoniae in neonatal unit of a teaching hospital in China from September 2015 to September 2016. Patients and methods We collected 12 carbapenem-resistant K. pneumoniae outbreak strains from 12 newborns and characterized these isolates for their antimicrobial susceptibility, clone relationships, and multi-locus sequence types using vitek-2 compact system, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Resistant genes were detected by using PCR and sequencing. Plasmid conjugation experiment was carried out to determine the transferability of carbapenem resistance. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and southern blotting were conducted for plasmid profiling. Results All 12 K. pneumoniae isolates were resistant to carbapenems and carried bla NDM-5, bla TEM-1 and bla SHV-11. Furthermore, PFGE analysis showed that NDM-5-producing K. pneumoniae were clonally related and MLST assigned them to sequence type 337. Conjugative assays showed that plasmids harboring bla NDM-5 gene were self-transmissible. Plasmid analysis suggested that all bla NDM-5 gene located on a ~45 kb IncX3 type plasmid. Conclusion To the best of our knowledge, this is the first report of a clone outbreak of bla NDM-5-carrying K. pneumoniae isolates from neonates. There is an urgent need for effective infection control measures to prevent bla NDM-5 variants from becoming epidemic in the neonates in the future.

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Haiquan Kang

Papers

SOS response and its regulation on the fluoroquinolone resistance.

High Prevalence of blaNDM Variants Among Carbapenem-Resistant Escherichia coli in Northern Jiangsu Province, China.

Performance of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry system for rapid identification of streptococci: a review.

Novel mutations in quinolone resistance-determining regions of gyrA, gyrB, parC and parE in Shigella flexneri clinical isolates from eastern Chinese populations between 2001 and 2011.

First Reported Nosocomial Outbreak Of NDM-5-Producing Klebsiella pneumoniae In A Neonatal Unit In China.