Environmental and Pathogenic Carbapenem Resistant Bacteria Isolated from a Wastewater Treatment Plant Harbour Distinct Antibiotic Resistance Mechanisms

Wastewater treatment plants are important reservoirs and sources for the dissemination of antibiotic resistance into the environment. Here, two different groups of carbapenem resistant bacteria—the potentially environmental and the potentially pathogenic—were isolated from both the wastewater influent and discharged effluent of a full-scale wastewater treatment plant and characterized by whole genome sequencing and antibiotic susceptibility testing. Among the potentially environmental isolates, there was no detection of any acquired antibiotic resistance genes, which supports the idea that their resistance mechanisms are mainly intrinsic. On the contrary, the potentially pathogenic isolates presented a broad diversity of acquired antibiotic resistance genes towards different antibiotic classes, especially β-lactams, aminoglycosides, and fluoroquinolones. All these bacteria showed multiple β-lactamase-encoding genes, some with carbapenemase activity, such as the blaKPC-type genes found in the Enterobacteriaceae isolates. The antibiotic susceptibility testing assays performed on these isolates also revealed that all had a multi-resistance phenotype, which indicates that the acquired resistance is their major antibiotic resistance mechanism. In conclusion, the two bacterial groups have distinct resistance mechanisms, which suggest that the antibiotic resistance in the environment can be a more complex problematic than that generally assumed.

Distinctive Roles of Two Acinetobactin Isomers in Challenging Host Nutritional Immunity

Acinetobacter baumannii has acquired antibiotic resistance at an alarming rate, and it is becoming a serious threat to society, particularly due to the paucity of effective treatment options. Acinetobactin is a siderophore of Acinetobacter baumannii , responsible for active iron supply, and it serves as a key virulence factor to counter host nutritional immunity during infection.

The distribution of antibiotic resistance genes in chicken gut microbiota commensals

Antibiotic resistance in bacterial pathogens or several indicator bacteria is commonly studied but the extent of antibiotic resistance in bacterial commensals colonising the intestinal tract is essentially unknown. In this study, we aimed to investigate the presence of horizontally acquired antibiotic resistance genes among chicken gut microbiota members in 259 isolates with known whole genomic sequences. Altogether 124 isolates contained at least one gene coding for antibiotic resistance. Genes coding for the resistance to tetracyclines (detected in 101 isolates), macrolide-lincosamide-streptogramin B antibiotics (28 isolates) and aminoglycosides (25 isolates) were the most common. The most frequent tetracycline resistance genes were tet(W), tet(32), tet(O) and tet(Q). Lachnospiraceae and Ruminococcaceae frequently encoded tet(W). Lachnospiraceae commonly coded also for tet(32) and tet(O). The tet(44) gene was associated with Erysipelotrichaceae and tet(Q) was detected in the genomes of Bacteroidaceae and Porphyromonadaceae. Without any bias we have shown that antibiotic resistance is quite common in gut commensals. However, a comparison of codon usage showed that the above-mentioned families represent the most common current reservoirs but probably not the original host of the detected resistances.

Pseudomonas aeruginosa Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters

Pseudomonas aeruginosa lung infection is difficult to eradicate due to the multiple (intrinsic and acquired) antibiotic resistance of bacteria and to their ability to produce a thick biofilm. Antibiotic treatment is hampered by poor antibiotic diffusion, efflux pump overexpression and the development of a persistent subpopulation with low metabolic activity. This is a cause for special concern in Cystic Fibrosis (CF) patients, where P. aeruginosa lung infection is the chief cause of morbidity and mortality. Combined tobramycin-ciprofloxacin treatment is routinely adopted due to the low frequency of resistant strains and its ostensible ability to control the infection. Nevertheless, symptoms usually recur, mainly due to the antibiotic persisters, which are difficult to detect in routine cultural microbiological assays. This chapter describes the issues involved in the microbiological diagnosis of P. aeruginosa lung infection in CF patients and the possible role of subinhibitory antibiotic concentrations in persister development and infection recurrence.

Mechanisms for Development of Ciprofloxacin Resistance in a Clinical Isolate of Pseudomonas aeruginosa

Treatment of infections by Pseudomonas aeruginosa is difficult due to its high intrinsic and acquired antibiotic resistance. Upon colonization in the human hosts, P. aeruginosa accumulates genetic mutations that confer the bacterium antibiotic resistance and ability to better live in the host environment. Characterizing the evolutionary traits would provide important insights into the development of effective combinatory antibiotic therapies to cure P. aeruginosa infections. In this work, we performed a detailed analysis of the molecular mechanisms by which a clinical isolate (CSP18) yields a ciprofloxacin-resistant derivative (CRP42). Genomic DNA re-sequencing and RNAseq were carried out to compare the genomic mutational signature and transcriptional profiles between the two isolates. The results indicated that D87G mutation in GyrA, together with MexEF-OprN hyper-expression caused by F7S mutation in MexS, was responsible for the increased resistance to ciprofloxacin in the isolate CRP42. Further simulation of CRP42 by gene editing in CSP18 demonstrated that D87G mutation in GyrA rendered CSP18 a fourfold increase in minimum inhibitory concentration against ciprofloxacin, while F7S mutation in MexS conferred an additional eightfold increase. Our experimental results demonstrate for the first time that the clinically relevant F7S point mutation in MexS results in hyper-expression of the mexEF-oprN and thus confers P. aeruginosa resistance to ciprofloxacin.

An Integrated HOCl-Producing E-Scaffold Is Active Against Monomicrobial and Polymicrobial Biofilms

Oxidizing agents like hypochlorous acid (HOCl) have antimicrobial activity. We developed an integrated electrochemical scaffold or ‘e-scaffold’ that delivers a continuous low dose of HOCl aimed at targeting microbial biofilms without exceeding concentrations toxic to humans, as a prototype of a device being developed to treat wound infections in humans. In this work, we tested the device against 33 isolates of bacteria (including isolates with acquired antibiotic resistance) grown as in vitro biofilms, alongside 12 combinations of dual-species in vitro biofilms. Biofilms were grown on the bottoms of 12-well plates for 24 hours. An integrated e-scaffold was placed atop each biofilm and polarized at 1.5V for 1, 2 or 4 hours. HOCl was produced electrochemically by oxidizing chloride ions (Cl-) in solution to chlorine (Cl2); dissolved Cl2 spontaneously dissociates in water to produce HOCl. The cumulative concentration of HOCl produced at the working electrode in each well was estimated to be 7.89, 13.46, and 29.50 mM after 1, 2 and 4 hours of polarization, respectively. Four hours of polarization caused an average reduction of 6.13 log10 CFU/cm2 (±1.99 log10 CFU/cm2) of viable cell counts of mono-species biofilms and 5.53 log10 CFU/cm2 (±2.31 log10 CFU/cm2) for the 12 dual-species biofilms studied. The described integrated e-scaffold reduces viable bacterial cell counts in biofilms formed by an array of antibiotic-susceptible and -resistant bacteria alone and in combination.

Enterococci: An Important Nosocomial Pathogen

Enterococci, particularly Enterococcus faecalis and Enterococcus faecium, are an important cause of nosocomial infections and have become a major issue worldwide. Nosocomial infections due to vancomycin resistant Enterococci (VRE) occur frequently. A significant increase in prevalence of VRE has been reported recently in many countries. Enterococci are second most frequent cause of nosocomial urinary tract infection, bacteremia and infective endocarditis. They are also related to etiology of intra-abdominal an pelvic infections, gastrointestinal infections and oral infections. The ability of Enterococci to survive in adverse conditions, presence of virulence factors and possession of intrinsic and acquired antibiotic resistance traits poses a therapeutic challenge. Due to high level of multidrug resistance in VRE, Enterococcus has become an important organism in health based settings.

A Pilot Study in Sweden on Efficacy of Benzylpenicillin, Oxytetracycline, and Florfenicol in Treatment of Acute Undifferentiated Respiratory Disease in Calves

Bovine respiratory disease (BRD) is a major indication for antibiotic treatment of cattle worldwide and some of the antibiotics used belong to classes of highest priority among those listed by WHO as critically important for human medicine. To preserve the efficacy of “newer” antibiotics, it has been suggested that “older” drugs should be revisited and used when possible. In this pilot study, we evaluated the efficacy of benzylpenicillin (PEN), oxytetracycline (OTC), and florfenicol (FLO) for treatment of naturally occurring BRD on two farms raising calves for slaughter. Farm personnel selected calves for enrolment, assigned calves to one of the three regimens in a systematically random manner, treated the calves, and registered the results. Overall, 117 calves were enrolled in the study. Nineteen calves relapsed in BRD before slaughter and were retreated (16.2%) and three died (2.6%). For PEN, treatment response rates after 30 days, 60 days, and until slaughter were 90.2%, 87.8%, and 80.5%, respectively; for OTC, 90.0%, 85.0%, and 85.0%, respectively; and for FLO, 86.1%, 83.3%, and 77.8%, respectively. There were no statistically significant differences in relapse, mortality, or response rates between the three treatment regimens. This indicates that PEN, OTC, and FLO were equally effective for treatment of BRD but the results need to be confirmed in a more elaborate study with a higher statistical power. The findings support the current recommendations from the Swedish Veterinary Association and the Medical Products Agency to use benzylpenicillin as a first line antibiotic for treatment of calves with undifferentiated respiratory disease in Sweden. Due to differences in the panorama of infectious agents and presence of acquired antibiotic resistance, the findings might not be applicable in other geographical areas.

Aminoglycosides: From Antibiotics to Building Blocks for the Synthesis and Development of Gene Delivery Vehicles

Aminoglycosides are a class of naturally occurring and semi synthetic antibiotics that have been used for a long time in fighting bacterial infections. Due to acquired antibiotic resistance and inherent toxicity, aminoglycosides have experienced a decrease in interest over time. However, in the last decade, we are seeing a renaissance of aminoglycosides thanks to a better understanding of their chemistry and mode of action, which had led to new trends of application. The purpose of this comprehensive review is to highlight one of these new fields of application: the use of aminoglycosides as building blocks for the development of liposomal and polymeric vectors for gene delivery. The design, synthetic strategies, ability to condensate the genetic material, the efficiency in transfection, and cytotoxicity as well as when available, the antibacterial activity of aminoglycoside-based cationic lipids and polymers are covered and critically analyzed.

Metagenomic analysis of acquired antibiotic resistance determinants in the gut microbiota of wild boars (Sus scrofa) – preliminary results

IntroductionLand application of manure that contains antibiotics and resistant bacteria may facilitate the establishment of an environmental reservoir of antibiotic-resistant microbes, promoting their dissemination into agricultural and natural habitats. The main objective of this study was to search for acquired antibiotic resistance determinants in the gut microbiota of wild boar populations living in natural habitats.Material and MethodsGastrointestinal samples of free-living wild boars were collected in the Zemplén Mountains in Hungary and were characterised by culture-based, metagenomic, and molecular microbiological methods. Bioinformatic analysis of the faecal microbiome of a hunted wild boar from Japan was used for comparative studies. Also, shotgun metagenomic sequencing data of two untreated sewage wastewater samples from North Pest (Hungary) from 2016 were analysed by bioinformatic methods. Minimum spanning tree diagrams for seven-gene MLST profiles of 104 E. coli strains isolated in Europe from wild boars and domestic pigs were generated in Enterobase.ResultsIn the ileum of a diarrhoeic boar, a dominant E. coli O112ab:H2 strain with intermediate resistance to gentamicin, tobramycin, and amikacin was identified, displaying sequence type ST388 and harbouring the EAST1 toxin astA gene. Metagenomic analyses of the colon and rectum digesta revealed the presence of the tetQ, tetW, tetO, and mefA antibiotic resistance genes that were also detected in the gut microbiome of four other wild boars from the mountains. Furthermore, the tetQ and cfxA genes were identified in the faecal microbiome of a hunted wild boar from Japan.ConclusionThe gastrointestinal microbiota of the free-living wild boars examined in this study carried acquired antibiotic resistance determinants that are highly prevalent among domestic livestock populations.