Development of a plasmid stabilization system in Vibrio natriegens for the high production of 1,3-propanediol and 3-hydroxypropionate

Vibrio natriegens is a promising industrial chassis with a super-fast growth rate and high substrate uptake rates. V. natriegens was previously engineered to produce 1,3-propanediol (1,3-PDO) from glycerol by overexpressing the corresponding genes in a plasmid. However, antibiotic selection pressure for plasmid stability was not satisfactory and plasmid loss resulted in reduced productivity of the bioprocess. In this study, we developed an antibiotic-free plasmid stabilization system for V. natriegens. The system was achieved by shifting the glpD gene, one of the essential genes for glycerol degradation, from the chromosome to plasmid. With this system, engineered V. natriegens can stably maintain a large expression plasmid during the whole fed-batch fermentation and accumulated 69.5 g/L 1,3-PDO in 24 h, which was 23% higher than that based on antibiotic selection system. This system was also applied to engineering V. natriegens for the production of 3-hydroxypropionate (3-HP), enabling the engineered strain to accumulate 64.5 g/L 3-HP in 24 h, which was 30% higher than that based on antibiotic system. Overall, the developed strategy could be useful for engineering V. natriegens as a platform for the production of value-added chemicals from glycerol. Graphic Abstract

Molecular detection of Mycoplasma pneumoniae in Community acquired pneumonia

Community acquired pneumonia (CAP) remains a common and serious illness despite availability of potent anti-microbials and effective vaccine. Two types of CAP are commonly recognised - typical and atypical. Typical pneumonia is usually caused by bacteria such as Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catharralis, whereas, atypical pneumonia can be caused by Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneuomophila. Mycoplasma pneumoniae is found to be the most common cause of CAP among atypical pathogens and is called “Walking pneumonia”. It is a common atypical respiratory pathogen that produces diseases of varied severity ranging from mild upper respiratory tract infection to severe atypical pneumonia and is also responsible for producing a wide spectrum of non-pulmonary manifestations like neurological, hepatic, cardiac diseases, hemolytic anemia, polyarthritis and erythema multiforme which occur in as many as 25% of infected persons. As it lacks cell wall they are resistant to β lactam antibiotics, hence accurate and rapid diagnosis of M. pneumoniae infections is critical to initiate appropriate antibiotic treatment. Identification of M pneumoniae allows narrowing of initial empirical regimen which decreases antibiotic selection pressure and may lessen the risk of resistance. In view of this present study will be conducted in a tertiary care hospital for identification of M pneumoniae in cases of CAP by Polymerase chain reaction (PCR). 1. To detect proportion of Mycoplasma Pneumoniae among cases of CAP. 2. Detection of 16SrRNA Mycoplasma pneumoniae by PCR.Clinico-radiologically diagnosed 92 CAP patients were included in the study. Out of which 15 cases were caused by typical CAP pathogens like Streptococcus species, K.pneumoniae & M.tuberculosis. Samples (Sputum Bronchoalveolar lavages) from 77 suspected cases of atypical pneumonia are inoculated on PPLO broth (Difco) followed by identification of genus specific 16S rRNA Mycoplasma pneumoniae using PCR. PCR was found to be positive in 9 (11.68%) out of 77 CAP patients. PCR was found to be positive in 9 (11.68%) out of 77 CAP patients. Detection of M.pneumoniae is essential for prompt diagnosis and start of empirical therapy, thereby reducing antibiotic selection pressure.

Transferable blaCTX-M Carrying Multidrug Resistant Escherichia coli from Pig Population of North Eastern Region of India

Background: We investigated the occurrence of blaCTX-M carrying extended spectrum beta lactamase (ESBL) producing Escherichia coli in pigs from 8 North-eastern states of India with special emphasis on the transferability of ESBL gene from resistant E. coli strains to the susceptible Salmonella strains by in vitro and in vivo.Methods: Fecal samples (n=790) were collected from pigs reared under organized and unorganized farming set up of entire North-eastern region of India. All the samples were processed for isolation and identification of E. coli. All the isolates were subjected to antimicrobial sensitivity assay by disc diffusion method followed by determination of ESBLs producing ability by double disc synergy test (DDST). All the ESBLs producing isolates were screened for blaCTX-M gene by PCR using specific primers. The representative blaCTX-M gene positive isolates were used as donor to determine the ability to transfer of resistance gene in Salmonella by in vitro and in vivo assays with and without antibiotic selection pressure. Result: A total of 2,291 E. coli was isolated, of which 1113 and 1178 were from organized and unorganized farms, respectively. Majority of the isolates were multi-drug resistant with highest resistance against amoxicillin (84.81%) followed by cefalexin (77.17%), sulphafurazole (56.79%), piperacillin (46.40%), tetracycline (38.29%) and cefexime (35.66%). Isolates from unorganized farms showed higher resistance than the isolates recovered from organized farms. A total of 654 (28.55%) isolates were confirmed as ESBL producers by double disc synergy test (DDST) method, of which 65 (2.84%) isolates were positive for blaCTX-M gene. Genotypically, isolates with specific amino acids substitution revealed variation in their antibiotic susceptibility by phenotypic method. blaCTX-M gene could be successfully transferred horizontally from E. coli (donor) to Salmonella (recipient) by in vitro (3.6±2.07x10-8 to 4.4±2.88x10-8 transconjugate per donor) and in vivo method. By in vivo method in pig model, the frequency of transfer was higher under the antibiotic selection pressure (6.6±3.05x10-5 to 7.2±1.92x10-5 trans-conjugants per donor) than without antibiotic pressure (5.6±2.3x10-4 to 6.8±3.35x10-4 trans-conjugants per donor).

A framework for identifying the recent origins of mobile antibiotic resistance genes

Since the introduction of antibiotics as therapeutic agents, many bacterial pathogens have developed resistance to antibiotics. Mobile resistance genes, acquired through horizontal gene transfer, play an important role in this process. Understanding from which bacterial taxa these genes were mobilized, and whether their origin taxa share common traits, is critical for predicting which environments and conditions contribute to the emergence of novel resistance genes. This knowledge may prove valuable for limiting or delaying future transfer of novel resistance genes into pathogens. The literature on the origins of mobile resistance genes is scattered and based on evidence of variable quality. Here, we summarize, amend and scrutinize the evidence for 37 proposed origins of mobile resistance genes. Using state-of-the-art genomic analyses, we supplement and evaluate the evidence based on well-defined criteria. Nineteen percent of reported origins did not fulfill the criteria to confidently assign the respective origin. Of the curated origin taxa, >90% have been associated with infection in humans or domestic animals, some taxa being the origin of several different resistance genes. The clinical emergence of these resistance genes appears to be a consequence of antibiotic selection pressure on taxa that are permanently or transiently associated with the human/domestic animal microbiome.

Expansion of a Subset Within the C2 Subclade of Escherichia coli Sequence Type 131 (ST131) Is Driving the Increasing Rates of Aminoglycoside Resistance

Background Sequence type 131 (ST131) of Escherichia coli is a pandemic clone that drives the increasing rates of antibiotic resistance. While the pervasiveness of ST131 clade C, especially subclades C2 and C1-M27, has been demonstrated in numerous global surveys, no report about the ST131 clades and their virotypes has been published from Iran so far. Methods A collection of 73 consecutive ST131 isolates from extraintestinal specimens was investigated for determination of virotypes, antibiotic susceptibility patterns, resistance/virulence determinants, and clade subsets. Results Most of the isolates belonged to subclade C2 (33/73; 45.2%), which had the highest virulence factor (VF) scores and resistance rates, followed by C1-M27 (18; 24.6%), C1-non-M27 (14; 19.1%), and A (8; 10.9%). The distinctive profiles of subclade C2 virulence genes were revealed by principle coordinates analysis testing. The distribution of the hlyA virulence gene among subclade C2 was not uniform, so that positive strains (21; 63.6%) showed significantly higher rates of resistance (blaCTX-M-15, blaOXA-1, aac(6’)-Ib-cr, aac(6’)-Ib, aac(3)-IIa) and virulence (hra, tia/hek, K5, cnf, papGII, papC) markers and gentamicin/tobramycin resistance. Virotype C as the most common virotype (34; 46.5%) was predominant among the subclade C1 population, while virotypes E and F (21; 28.7%) were detected among subclade C2, which had the highest VF scores and aminoglycoside resistance rates. Conclusions The appearance of virotypes E and F among subclade C2 strains with higher rates of aminoglycoside resistance/virulence gene content shows the shifting dynamics of this pandemic clone in response to antibiotic selection pressure by establishing subsets with higher survival potential.

Antecedent Carbapenem Exposure as a Risk Factor for Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae and Carbapenemase-Producing Enterobacteriaceae

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) can be mechanistically classified into carbapenemase-producing Enterobacteriaceae (CPE) and non-carbapenemase-producing carbapenem nonsusceptible Enterobacteriaceae (NCPCRE). We sought to investigate the effect of antecedent carbapenem exposure as a risk factor for NCPCRE versus CPE. Among all patients with CRE colonization and infection, we conducted a case-control study comparing patients with NCPCRE (cases) and patients with CPE (controls). The presence of carbapenemases was investigated with phenotypic tests followed by PCR for predominant carbapenemase genes. We included 843 unique patients with first-episode CRE, including 387 (45.9%) NCPCRE and 456 (54.1%) CPE. The resistance genes detected in CPEs were blaNDM (42.8%), blaKPC (38.4%), and blaOXA-48-like (12.1%). After adjusting for confounders and clustering at the institutional level, the odds of prior 30-day carbapenem exposure was three times higher among NCPCRE than CPE patients (adjusted odds ratio [aOR], 3.48; 95% confidence interval [CI], 2.39 to 5.09; P < 0.001). The odds of prior carbapenem exposure and NCPCRE detection persisted in stratified analyses by Enterobacteriaceae species (Klebsiella pneumoniae and Escherichia coli) and carbapenemase gene (blaNDM and blaKPC). CPE was associated with male gender (aOR, 1.45; 95% CI, 1.07 to 1.97; P = 0.02), intensive care unit stay (aOR, 1.84; 95% CI, 1.24 to 2.74; P = 0.003), and hospitalization in the preceding 1 year (aOR, 1.42; 95% CI, 1.01 to 2.02; P = 0.05). In a large nationwide study, antecedent carbapenem exposure was a significant risk factor for NCPCRE versus CPE, suggesting a differential effect of antibiotic selection pressure.

Hygiene Hampers Competitive Release of Resistant Bacteria in the Commensal Microbiota

Good hygiene, in both health care and the community, is central to containing the rise of antibiotic resistance, as well as to infection control more generally. But despite the well-known importance, the ecological mechanisms by which hygiene affects resistance evolution remain obscure. Using metacommunity ecology theory, we here propose that hygiene attenuates the effect of antibiotic selection pressure. Specifically, we predict that hygiene limits the scope for antibiotics to induce competitive release of resistant bacteria within treated hosts, and that this is due to a modulating effect of hygiene on the distribution of resistant and sensitive strains in the host population. We show this in a mathematical model of bacterial metacommunity dynamics, and test the results against data on antibiotic resistance, antibiotic treatment, and the use of alcohol-based hand rub in long-term care facilities. Our results underscore the importance of hygiene, and point to a concrete way to weaken the link between antibiotic use and increasing resistance.

Epidemiology and molecular characterization of Enterobacteriaceae producing Extended spectrum β-lactamase in extensive and extensive breeding animals in Burkina Faso

Summary Background Antimicrobial resistance genes can be found in all ecosystems even in those where antibiotic selection pressure is less exerted. Extended spectrum 𝛽-lactamases (ESBL) determinants have been detected in clinical isolates and commensal bacteria from humans and animals. Here we investigated, for the first time, the ESBL-producing Enterobacteriaceae in stool from intensive and extensive breeding animals (cattle, pigs and poultry) in Burkina Faso. Results from March to June 2017 in the Bobo Dioulasso area, we investigated stool samples collected from healthy animals (cattle = 251; pigs = 250 and poultry = 397) in one (1) slaughterhouse, five (5) livestock farms and one (1) poultry market. The frequency of ESBL genes carriage was 41.03% among cattle, 69.60% among pigs, 0.8% among intensive farming and 19.1% among extensive poultry farming. Only all the poultry were fed with antibiotics. The bacterial strains carrying the ESBL were E. coli (278/315) and K. pneumoniae (36/315). The ESBL genes carried were CTX-M 15, TEM and Oxa-1-like. These three β- lactamase genes were associated in some bacterial strains. The E. coli strains belonged most commonly to the phylogroup A. Conclusion This study showed the high resistance of Enterobacteriaceae to antibiotics in livestock in Burkina Faso by production of ESBL. This high level of resistance was observed in animals that did not receive antibiotics. This situation could suggest an environmental contamination of the livestock with ESBL-producing bacteria.