The Diversity, Resistance Profiles and Plasmid Content of Klebsiella spp. Recovered from Dairy Farms Located around Three Cities in Pakistan.

The rise of antimicrobial resistance (AMR) in bacterial pathogens such as Klebsiella pneumoniae (Kp) is a pressing public health and economic concern. The 'One-Health' framework recognizes that effective management of AMR requires surveillance in agricultural as well as clinical settings, particularly in low-resource regions such as Pakistan. Here, we use whole-genome sequencing to characterise 49 isolates of Klebisella spp. (including 43 Kp) and 2 presumptive Providencia rettgeri isolates recovered from dairy farms located near 3 cities in Pakistan-Quetta (n = 29), Faisalabad (n = 19), and Sargodha (n = 3). The 43 Kp isolates corresponded to 38 sequence types (STs), and 35 of these STs were only observed once. This high diversity indicates frequent admixture and limited clonal spread on local scales. Of the 49 Klebsiella spp. isolates, 41 (84%) did not contain any clinically relevant antimicrobial resistance genes (ARGs), and we did not detect any ARGs predicted to encode resistance to carbapenems or colistin. However, four Kp lineages contained multiple ARGs: ST11 (n = 2), ST1391-1LV (n = 1), ST995 (n = 1) and ST985 (n = 1). STs 11, 1391-1LV and 995 shared a core set of five ARGs, including blaCTX-M-15, harboured on different AMR plasmids. ST985 carried a different set of 16 resistance genes, including blaCTX-M-55. The two presumptive P. rettgeri isolates also contained multiple ARGs. Finally, the four most common plasmids which did not harbour ARGs in our dataset were non-randomly distributed between regions, suggesting that local expansion of the plasmids occurs independently of the host bacterial lineage. Evidence regarding how dairy farms contribute to the emergence and spread of AMR in Pakistan is valuable for public authorities and organizations responsible for health, agriculture and the environment, as well as for industrial development.

A large-scale genomic snapshot of Klebsiella spp. isolates in Northern Italy reveals limited transmission between clinical and non-clinical settings.

The Klebsiella group, found in humans, livestock, plants, soil, water and wild animals, is genetically and ecologically diverse. Many species are opportunistic pathogens and can harbour diverse classes of antimicrobial resistance genes. Healthcare-associated Klebsiella pneumoniae clones that are non-susceptible to carbapenems can spread rapidly, representing a high public health burden. Here we report an analysis of 3,482 genome sequences representing 15 Klebsiella species sampled over a 17-month period from a wide range of clinical, community, animal and environmental settings in and around the Italian city of Pavia. Northern Italy is a hotspot for hospital-acquired carbapenem non-susceptible Klebsiella and thus a pertinent setting to examine the overlap between isolates in clinical and non-clinical settings. We found no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside the clinical environment. Although we noted occasional transmission between clinical and non-clinical settings, our data point to a limited role of animal and environmental reservoirs in the human acquisition of Klebsiella spp. We also provide a detailed genus-wide view of genomic diversity and population structure, including the identification of new groups.

Staphylococcus aureus lineages associated with a free-ranging population of the fruit bat Pteropus livingstonii retained over 25 years in captivity.

Conservation of endangered species has become increasingly complex, and costly interventions to protect wildlife require a robust scientific evidence base. This includes consideration of the role of the microbiome in preserving animal health. Captivity introduces stressors not encountered in the wild including environmental factors and exposure to exotic species, humans and antimicrobial drugs. These stressors may perturb the microbiomes of wild animals, with negative consequences for their health and welfare and hence the success of the conservation project, and ultimately the risk of release of non-native organisms into native ecosystems. We compared the genomes of Staphylococcus aureus colonising critically endangered Livingstone's fruit bats (Pteropus livingstonii) which have been in a captive breeding programme for 25 years, with those from bats in the endemic founder population free ranging in the Comoros Republic. Using whole genome sequencing, we compared 47 isolates from captive bats with 37 isolates from those free ranging in the Comoros Republic. Our findings demonstrate unexpected resilience in the bacteria carried, with the captive bats largely retaining the same two distinctive lineages carried at the time of capture. In addition, we found evidence of genomic changes which suggest specific adaptations to the bat host.

Challenges in realising the potential of wastewater-based epidemiology to quantitatively monitor and predict the spread of disease.

Researchers around the world have demonstrated correlations between measurements of SARS-CoV-2 RNA in wastewater (WW) and case rates of COVID-19 derived from direct testing of individuals. This has raised concerns that wastewater-based epidemiology (WBE) methods might be used to quantify the spread of this and other diseases, perhaps faster than direct testing, and with less expense and intrusion. We illustrate, using data from Scotland and the USA, the issues regarding the construction of effective predictive models for disease case rates. We discuss the effects of variation in, and the problem of aligning, public health (PH) reporting and WW measurements. We investigate time-varying effects in PH-reported case rates and their relationship to WW measurements. We show the lack of proportionality of WW measurements to case rates with associated spatial heterogeneity. We illustrate how the precision of predictions is affected by the level of aggregation chosen. We determine whether PH or WW measurements are the leading indicators of disease and how they may be used in conjunction to produce predictive models. The prospects of using WW-based predictive models with or without ongoing PH data are discussed.

Fatal exudative dermatitis in island populations of red squirrels (Sciurus vulgaris): spillover of a virulent Staphylococcus aureus clone (ST49) from reservoir hosts.

Fatal exudative dermatitis (FED) is a significant cause of death of red squirrels (Sciurus vulgaris) on the island of Jersey in the Channel Islands where it is associated with a virulent clone of Staphylococcus aureus, ST49. S. aureus ST49 has been found in other hosts such as small mammals, pigs and humans, but the dynamics of carriage and disease of this clone, or any other lineage in red squirrels, is currently unknown. We used whole-genome sequencing to characterize 228 isolates from healthy red squirrels on Jersey, the Isle of Arran (Scotland) and Brownsea Island (England), from red squirrels showing signs of FED on Jersey and the Isle of Wight (England) and a small number of isolates from other hosts. S. aureus was frequently carried by red squirrels on the Isle of Arran with strains typically associated with small ruminants predominating. For the Brownsea carriage, S. aureus was less frequent and involved strains associated with birds, small ruminants and humans, while for the Jersey carriage S. aureus was rare but ST49 predominated in diseased squirrels. By combining our data with publicly available sequences, we show that the S. aureus carriage in red squirrels largely reflects frequent but facile acquisitions of strains carried by other hosts sharing their habitat ('spillover'), possibly including, in the case of ST188, humans. Genome-wide association analysis of the ruminant lineage ST133 revealed variants in a small number of mostly bacterial-cell-membrane-associated genes that were statistically associated with squirrel isolates from the Isle of Arran, raising the possibility of specific adaptation to red squirrels in this lineage. In contrast there is little evidence that ST49 is a common carriage isolate of red squirrels and infection from reservoir hosts such as bank voles or rats, is likely to be driving the emergence of FED in red squirrels.

Genome of Superficieibacter maynardsmithii, a novel, antibiotic susceptible representative of Enterobacteriaceae.

During a citywide microbiological screening project in Pavia (Italy) a bacterial strain isolated from the surface of an Automated Teller Machine was classified as a Klebsiella sp. by MALDI-TOF spectrometry, and shown to be susceptible to the most antimicrobial classes by phenotypic testing. After Illumina genome sequencing and subsequent assembly, a high-quality draft genome was obtained (size = 5,051,593 bp, N50 = 615,571 bp, largest contig = 1,328,029 bp, N_contig = 17, GC content = 51.58%, coverage = 141.42), absence of antimicrobial resistance genes was confirmed, but the strain resulted to be highly divergent from all Klebsiella, and more related to other Enterobacteriaceae. The higher values of 16S rRNA identity were with members of the genera Citrobacter, Salmonella, and "Superficieibacter." An ortholog-based phylogenomic analysis indicated a sister group relationship with "Superficieibacter electus," in a distinct clade from other members of the Enterobacteriaceae family. In order to evaluate whether the novel genome represents a new species of "Superficiebacter," average nucleotide identity (ANI) and Hadamard analysis were performed on a dataset of 78 Enterobacteriaceae. The novel genome showed an ANI of 87.51% with S. electus, which compared on identity values between other members of the family, clearly indicates that the genome represents a new species within the genus "Superficieibacter." We propose for the new species the name "Superficieibacter maynardsmithii."

A high prevalence of blaOXA-48 in Klebsiella (Raoultella) ornithinolytica and related species in hospital wastewater in South West England.

Klebsiella species occupy a wide range of environmental and animal niches, and occasionally cause opportunistic infections that are resistant to multiple antibiotics. In particular, Klebsiella pneumoniae (Kpne) has gained notoriety as a major nosocomial pathogen, due principally to the rise in non-susceptibility to carbapenems and other beta-lactam antibiotics. Whilst it has been proposed that the urban water cycle facilitates transmission of pathogens between clinical settings and the environment, the level of risk posed by resistant Klebsiella strains in hospital wastewater remains unclear. We used whole genome sequencing (WGS) to compare Klebsiella species in contemporaneous samples of wastewater from an English hospital and influent to the associated wastewater treatment plant (WWTP). As we aimed to characterize representative samples of Klebsiella communities, we did not actively select for antibiotic resistance (other than for ampicillin), nor for specific Klebsiella species. Two species, Kpne and K. (Raoultella) ornithinolytica (Korn), were of equal dominance in the hospital wastewater, and four other Klebsiella species were present in low abundance in this sample. In contrast, despite being the species most closely associated with healthcare settings, Kpne was the dominant species within the WWTP influent. In total, 29 % of all isolates harboured the blaOXA-48 gene on a pOXA-48-like plasmid, and these isolates were almost exclusively recovered from the hospital wastewater. This gene was far more common in Korn (68 % of isolates) than in Kpne (3.4 % of isolates). In general plasmid-borne, but not chromosomal, resistance genes were significantly enriched in the hospital wastewater sample. These data implicate hospital wastewater as an important reservoir for antibiotic-resistant Klebsiella, and point to an unsuspected role of species within the Raoultella group in the maintenance and dissemination of plasmid-borne blaOXA-48. This article contains data hosted by Microreact.

Conformation control of the histidine kinase BceS of Bacillus subtilis by its cognate ABC-transporter facilitates need-based activation of antibiotic resistance.

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost-effective strategy is achieved via a two-component regulatory system (BceRS) working in a sensory complex with an ABC-transporter (BceAB), together acting as a flux-sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston-like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP-like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux-dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub-maximal sensitivity. These results provide the first mechanistic insights into transport flux-dependent signaling, a unique strategy for energy-efficient decision making.

Closed genome sequences of Staphylococcus lloydii sp. nov. and Staphylococcus durrellii sp. nov. isolated from captive fruit bats (Pteropus livingstonii).

The increasing availability of whole genome sequencing of bacteria has accelerated the discovery of novel species which may not have been easy to discriminate using standard phenotypic or single gene methods. Phylogenomic analysis of genome sequences from a collection of coagulase-negative staphylococcal species isolated from captive fruit bats revealed two clusters which were close to Staphylococcus kloosii. To assess the relatedness of the strains we used digital DNA-DNA hybridization (dDDH) and two methods for average nucleotide identity (ANI) computation which predicted two novel species having dDDH less than 70 % and ANI less than 95%. We propose these species as Staphylococcus lloydii sp. nov. (type strain 23_2_7_LYT=NCTC 14453T=DSM 111639T) and Staphylococcus durrellii sp. nov (type strain 27_4_6_LYT=NCTC 14454T=DSM 111640T).

Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons.

Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants. The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P. syringae pv. tomato strain PT23 and pAV505 from P. syringae pv. phaseolicola strain HRI1302A, were isolated. DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively. Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da. Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph). The predicted peptides showed an overall identity of 897 %, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa). The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other CoIE2-related plasmids. However, both peptides were 100 residues longer than any of the known CoIE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the ru/AB genes, involved in UV resistance, from plasmid pPSR1. It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids.