Broad-spectrum Antimicrobial Resistance Research Articles

Whole genome sequencing and genotyping Klebsiella pneumoniae multi-drug resistant hospital isolates from Western Kenya.

Klebsiella pneumoniae are a frequent cause of nosocomial infections worldwide. Sequence type 147 (ST147) has been reported as a major circulating high-risk lineage in many countries, and appears to be a formidable platform for the dissemination of antimicrobial resistance (AMR) determinants. However, the distribution of this pathogen in Western African hospitals has been scarcely studied. The main objective of this work was to perform whole genome sequencing of K. pneumoniae isolates from a referral hospital in Kakamega (Kenya) for genotyping and identification of AMR and virulence determinants. In total, 15 K. pneumoniae isolates showing a broad spectrum antimicrobial resistance were selected for whole genome sequencing by Illumina HiSeq 2500 platform. ST147 was the dominant lineage among the highly-resistant K. pneumoniae isolates that we sequenced. ST147 was associated with both community- and the hospital-acquired infections, and with different infection sites, whereas other STs were predominantly uropathogens. Multiple antibiotic resistance and virulence determinants were detected in the genomes including extended-spectrum β-lactamases (ESBL) and carbapenemases. Many of these genes were plasmid-borne. Our data suggest that the evolutionary success of ST147 may be linked with the acquisition of broad host-range plasmids, and their propensity to accrue AMR and virulence determinants. Although ST147 is a dominant lineage in many countries worldwide, it has not been previously reported as prevalent in Africa. Our data suggest an influx of new nosocomial pathogens with new virulence genes into African hospitals from other continents.

Characterization and multilocus sequence typing of Clostridium perfringens isolated from patients with diarrhoea and food poisoning in Tai'an region, China

ObjectivesClostridium perfringens (C. perfringens) is a significant opportunistic pathogen. This study aims to examine the occurrence of C. perfringens in patients with diarrhoea and food poisoning and compare the genetic similarities with strains found in poultry retail markets and poultry farms in the same city (Tai'an, China). MethodsClostridium perfringens was isolated from 30 human faecal samples and genotyped using multiplex PCR. The antimicrobial susceptibility test was conducted using the Kirby-Bauer disk diffusion method. Genetic relationships were analysed through Multi-locus sequence typing (MLST) and Phylogenetic analysis. ResultsThe positive rate of C. perfringens was found to be 96.67%. Among the positive samples, 91.67% of the faecal samples from patients with food poisoning contained type F strains of C. perfringens, while only 16.67% of the samples from diarrhoea cases contained type F. The drug susceptibility test revealed that the majority of isolates displayed broad-spectrum antimicrobial resistance. Out of the 57 isolates tested for drug susceptibility, 89.47% demonstrated resistance to at least three antibiotics. The MLST results indicated that strains originating from the same host and environment tended to be more closely related. However, certain strains associated with food poisoning and diarrhoea in patients shared the same ST and CC as some strains found in the retail market. These strains were also found to be phylogenetically similar to some retail market strains, suggesting potential risks to human health. ConclusionsTherefore, it is crucial to enhance the management of poultry retail markets in order to mitigate these associated risks.

Cloning, improved expression and purification of invasion plasmid antigen D (IpaD): an effector protein of enteroinvasive Escherichia coli (EIEC)

ABSTRACT The widespread increase in broad-spectrum antimicrobial resistance is making it more difficult to treat gastrointestinal infections. Enteroinvasive Escherichia coli is a prominent etiological agent of bacillary dysentery, invading via the fecal-oral route and exerting virulence on the host via the type III secretion system. IpaD, a surface-exposed protein on the T3SS tip that is conserved among EIEC and Shigella, may serve as a broad immunogen for bacillary dysentery protection. For the first time, we present an effective framework for improving the expression level and yield of IpaD in the soluble fraction for easy recovery, as well as ideal storage conditions, which may aid in the development of new protein therapies for gastrointestinal infections in the future. To achieve this, uncharacterized full length IpaD gene from EIEC was cloned into pHis-TEV vector and induction parameters were optimized for enhanced expression in the soluble fraction. After affinity-chromatography based purification, 61% pure protein with a yield of 0.33 mg per litre of culture was obtained. The purified IpaD was retained its secondary structure with a prominent α-helical structure as well as functional activity during storage, at 4°C, −20°C and −80°C using 5% sucrose as cryoprotectants, which is a critical criterion for protein-based treatments.

Probiotics at War Against Viruses: What Is Missing From the Picture?

Our world is now facing a multitude of novel infectious diseases. Bacterial infections are treated with antibiotics, albeit with increasing difficulty as many of the more common causes of infection have now developed broad spectrum antimicrobial resistance. However, there is now an even greater challenge from both old and new viruses capable of causing respiratory, enteric, and urogenital infections. Reports of viruses resistant to frontline therapeutic drugs are steadily increasing and there is an urgent need to develop novel antiviral agents. Although this all makes sense, it seems rather strange that relatively little attention has been given to the antiviral capabilities of probiotics. Over the years, beneficial strains of lactic acid bacteria (LAB) have been successfully used to treat gastrointestinal, oral, and vaginal infections, and some can also effect a reduction in serum cholesterol levels. Some probiotics prevent gastrointestinal dysbiosis and, by doing so, reduce the risk of developing secondary infections. Other probiotics exhibit anti-tumor and immunomodulating properties, and in some studies, antiviral activities have been reported for probiotic bacteria and/or their metabolites. Unfortunately, the mechanistic basis of the observed beneficial effects of probiotics in countering viral infections is sometimes unclear. Interestingly, in COVID-19 patients, a clear decrease has been observed in cell numbers of Lactobacillus and Bifidobacterium spp., both of which are common sources of intestinal probiotics. The present review, specifically motivated by the need to implement effective new counters to SARS-CoV-2, focusses attention on viruses capable of co-infecting humans and other animals and specifically explores the potential of probiotic bacteria and their metabolites to intervene with the process of virus infection. The goal is to help to provide a more informed background for the planning of future probiotic-based antiviral research.

Next-Generation Sequencing of the Whole Bacterial Genome for Tracking Molecular Insight into the Broad-Spectrum Antimicrobial Resistance of Helicobacter pylori Clinical Isolates from the Democratic Republic of Congo.

Antimicrobial susceptibility testing (AST) is increasingly needed to guide the Helicobacter pylori (H. pylori) treatment but remains laborious and unavailable in most African countries. To assess the clinical relevance of bacterial whole genome sequencing (WGS)-based methods for predicting drug susceptibility in African H. pylori, 102 strains isolated from the Democratic Republic of Congo were subjected to the phenotypic AST and next-generation sequencing (NGS). WGS was used to screen for the occurrence of genotypes encoding antimicrobial resistance (AMR). We noted the broad-spectrum AMR of H. pylori (rates from 23.5 to 90.0%). A WGS-based method validated for variant discovery in AMR-related genes (discovery rates of 100%) helped in identifying mutations of key genes statistically related to the phenotypic AMR. These included mutations often reported in Western and Asian populations and, interestingly, several putative AMR-related new genotypes in the pbp1A (e.g., T558S, F366L), gyrA (e.g., A92T, A129T), gyrB (e.g., R579C), and rdxA (e.g., R131_K166del) genes. WGS showed high performance for predicting AST phenotypes, especially for amoxicillin, clarithromycin, and levofloxacin (Youden’s index and Cohen’s Kappa > 0.80). Therefore, WGS is an accurate alternative to the phenotypic AST that provides substantial decision-making information for public health policy makers and clinicians in Africa, while providing insight into AMR mechanisms for researchers.

Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China

Clostridium perfringens is an important zoonotic microorganism, which can cause diseases in animal and human under certain conditions. Contamination of C. perfringens in chicken and pork meat has been reported worldwide, but it is rarely reported in duck products. The current study was undertaken to investigate C. perfringens contamination in duck products from a large retail market in Tai'an region, China and the serotype distribution, antimicrobial resistance and genetic relatedness of the isolates. In total, 173 samples of duck products, 10 samples of environmental origins and 7 samples of fresh faeces from healthy shopkeepers were collected between March and November 2018, of which, 58 (31.69%), 10 (100%) and 7 (100%) samples were determined to be positive for C. perfringens, respectively. Ninety-nine isolates of C. perfringens were recovered, all of which were identified as type A. Beta2 (cpb2) toxin gene was found in 54.30% and 33.30% of the isolates from duck products and healthy shopkeepers, respectively. Antimicrobial susceptibility testing revealed that 90.10% of the isolates from duck products and environment showed multiple antibiotic resistance, among which, 49.40% were resistant to at least 6 classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 58 representative isolates were divided into 41 sequences types (STs), among which, ST11 (8.60%) was the most common; 37.90% of all isolates were classified into four clonal complexes (CC1-CC4). The most prolific clonal complex (CC1), accounting for 24.13% of all isolates, contained isolates mainly from carcass, animal intestinal contents and environment of four retail stores. A portion of human isolates and duck isolates was distributed in the same CC or ST. In conclusion, C. perfringens contamination in some duck products in Tai'an retail market was relatively high, and most of the isolates exhibited broad-spectrum antimicrobial resistance. Although all the isolates belong to type A, considerable genetic diversity was observed, and a portion of the strains from human and duck was found to be phylogenetically close. The results indicated that antimicrobial-resistance strains of duck origin pose a potential threat to humans by spreading through the food chain.

Antibacterial activity of ceftolozane/tazobactam alone and in combination with other antimicrobial agents against MDR Pseudomonas aeruginosa.

Broad-spectrum antimicrobial resistance in Pseudomonas aeruginosa (PSA) isolates is a growing concern as our therapeutic options have become significantly limited. Although ceftolozane/tazobactam (C/T) has been shown to be highly active against MDR PSA pathogens, combination regimens are often employed in real-world settings. To assist the clinical decision-making process regarding the selection of combination antibiotics and dosages for this pathogen, we performed time-kill studies assessing clinical free peak and trough C/T concentrations alone and in combination with eight anti-pseudomonal agents against four clinical MDR PSA isolates. Time-kill analyses were performed over 24 h in duplicate using C/T concentrations reflective of the free peak concentrations of a 3 g dose every 8 h (q8h; 120/25.2 mg/L) and the peak and trough of a 1.5 g q8h dose (60/12.6 and 7.5/1.6 mg/L) in humans. The activity of C/T 120, 60 and 7.5 mg/L alone and C/T 7.5 mg/L in combination with free peak and trough concentrations of clinical doses for cefepime, ciprofloxacin, colistin, aztreonam, meropenem, piperacillin/tazobactam, fosfomycin and amikacin was tested for all isolates. C/T 3 and 1.5 g q8h peak concentrations demonstrated killing against the MDR PSA. Colistin and fosfomycin were synergistic with C/T as dual therapy and triple therapy regimens. As a result of escalating resistance, PSA is an increasingly challenging pathogen in the clinical setting. Our findings aid in the identification of novel treatment options using achievable drug exposures for the treatment of MDR PSA.

Prevalence and Antimicrobial Resistance of Salmonella Isolates Recovered from Retail Pork in Major Village Markets in Tai'an Region, China

The current study was undertaken to evaluate Salmonella contamination in retail pork at major village markets of the Tai'an region, China. In total, 200 retail pork samples were collected from four village markets between June 2015 and February 2016, of which 69 samples (34.5%) were determined to be positive for Salmonella. Eleven serotypes were identified from the 69 Salmonella isolates, and Salmonella Derby was the most common (18 of 69, 26.1%), followed by Typhimurium (17 of 69, 24.6%) and Meleagridis (11 of 69, 15.9%). Antimicrobial susceptibility testing showed that antimicrobial resistance against tetracycline was the most prevalent (42 of 69, 60.9%), but antimicrobial resistance against both ceftriaxone and cefotaxime was 1.4% (1 of 69) and 2.9% (2 of 69), respectively. Multilocus sequence typing revealed that the 69 Salmonella isolates were divided into 11 sequence types (STs), among which ST40 (18 of 69, 26.1%) was the most common, followed by ST34 (15 of 69, 21.7%) and ST64 (13 of 69, 18.8%). Collectively, retail pork at village markets in the Tai'an region has a high Salmonella contamination rate, and these isolates exhibit broad-spectrum antimicrobial resistance. However, the absence of a dominant ST demonstrates that the Salmonella isolates from retail pork may be of diverse origins.

A Multilocus Sequence Typing Scheme Implies Population Structure and Reveals Several Putative Novel Achromobacter Species

The genus Achromobacter currently is comprised of seven species, including Achromobacter xylosoxidans, an opportunistic and nosocomial pathogen that displays broad-spectrum antimicrobial resistance and is recognized as causing chronic respiratory tract infection in persons with cystic fibrosis (CF). To enable strain typing for global epidemiologic investigations, to clarify the taxonomy of "Achromobacter-like" strains, and to elucidate the population structure of this genus, we developed a genus-level multilocus sequence typing (MLST) scheme. We employed in silico analyses of whole-genome sequences of several phylogenetically related genera, including Bordetella, Burkholderia, Cupriavidus, Herminiimonas, Janthinobacterium, Methylibium, and Ralstonia, for selecting loci and designing PCR primers. Using this MLST scheme, we analyzed 107 genetically diverse Achromobacter isolates cultured from biologic specimens from CF and non-CF patients, 1 isolate recovered from sludge, and an additional 39 strains obtained from culture collections. Sequence data from these 147 strains, plus three recently genome-sequenced Achromobacter strains, were assigned to 129 sequence types based on seven loci. Calculation of the nucleotide divergence of concatenated locus sequences within and between MLST clusters confirmed the seven previously named Achromobacter species and revealed 14 additional genogroups. Indices of association showed significant linkage disequilibrium in all of the species/genogroups able to be tested, indicating that each group has a clonal population structure. No clear segregation of species/genogroups between CF and non-CF sources was found.

Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

Chryseobacterium respiratory tract infections in patients with cystic fibrosis

To investigate the prevalence of infections by Chryseobacterium in a cohort of cystic fibrosis (CF) patients, to assess the antimicrobial susceptibility of these strains, to examine their DNA fingerprinting and to evaluate some clinical outcomes of patients infected by these bacteria. Patients (300) attending a Regional Cystic Fibrosis Unit were enrolled in this study over 4 years. Natural or induced sputum samples were processed for microscopic and cultural tests. For phenotypic identification, automated and manual systems were used. For chemosusceptibility test, an automatic broth microdilution test and a disk-diffusion test were used and strains underwent DNA fingerprinting by pulsed-field gel electrophoresis (PFGE). Thirty-five strains of Chryseobacterium were isolated from 22 patients. These strains showed a broad-spectrum antimicrobial resistance, with activity only for trimethoprim-sulfamethoxazole and quinolones. PFGE profiles of all isolates were generally heterogeneous, suggesting independent circulation. This is the first report about clinical isolates of Chryseobacterium spp from CF patients in an Italian Centre. The infection by Chryseobacterium was not associated to a deterioration of pulmonary function and mortality: therefore, all patients infected by Chryseobacterium were co-infected by Pseudomonas aeruginosa and 3 of these were also co-infected by Burkholderia cepacia complex.

Burkholderiaand Emerging Pathogens in Cystic Fibrosis

Persons with cystic fibrosis (CF) are susceptible to respiratory tract infection with a variety of bacterial species. Among these are several species in the genus Burkholderia (including members of the B. cepacia complex, B. gladioli, and B. pseudomallei), Stenotrophomonas maltophilia, Achromobacter ( Alcaligenes) xylosoxidans, certain Ralstonia species, and species within the new genus Pandoraea. Enterobacteriaceae and several other rather unusual or novel species also can be found. Many of these species are closely related phylogenetically and have similar phenotypes, presenting a challenge to accurate laboratory identification. Although their role in contributing to pulmonary disease in CF is not clear, the incidence of infection by some of these species appears to be increasing. The broad-spectrum antimicrobial resistance typically exhibited by these species severely limits treatment options. Newly developed genotypic methods of identification will enable studies to better assess the epidemiology and natural history of these emerging pathogens in CF.

Waste not, want not

A depressingly large proportion of agricultural production (US$30–50 billion per annum) is lost to plant pathogens. Although genetically engineered plants have been developed with anti-microbial properties, until now the spectrum of such protection has been relatively narrow. Milan Osusky and colleagues [Nat. Biotechnol. (2000) 18, 1162–1166] report the introduction of broad-spectrum antimicrobial resistance to both bacterial and fungal pathogens into potato, one of the world's major food crops using genetic engineering. However, although this has promise for developing disease-resistant plants, the benefits might be potato-cultivar-specific. NC