Genomic evidence of environmental and resident Salmonella Senftenberg and Montevideo contamination in the pistachio supply-chain

Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed an in-depth retrospective data analysis of Salmonella associated with pistachios as well as a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed utilizing short-read and long-read sequencing technologies. The sequence data were analyzed using two methods: the FDA’s Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multilocus Sequence Typing (wgMLST). The year-long storage study evaluated the survival of five strains of Salmonella on pistachios stored at 25 °C at 35% and 54% relative humidity (RH). Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) the use of metagenomic analysis is a novel tool for determining the composition of serovar survival in a cocktail inoculated storage study.

Complete Circular Genome Sequence of a mecB- and mecD -Containing Strain of Macrococcus canis

The complete genome sequence of Macrococcus canis strain 19/EPI0118, isolated from a veterinary clinic environment in Switzerland, was determined using hybrid assembly of Oxford Nanopore and Illumina reads. 19/EPI0118 harbored the methicillin resistance genes mecB and mecD on a staphylococcal cassette chromosome mec element and a Macrococcus chromosomal resistance island, respectively.

Vegetable-derived carbapenemase-producing high-risk Klebsiella pneumoniae ST15 and Acinetobacter baumannii ST2 clones in Japan: co-existence of blaNDM-1, blaOXA-66, blaOXA-72, and AbaR4-like resistance island from the same sample

This study was conducted to characterize carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumannii isolated from fresh vegetables in Japan. Two K. pneumoniae (AO15, and AO22) and one A. baumannii (AO22) isolates collected from vegetables in the Higashihiroshima city, Japan and subjected to antimicrobial susceptibility testing, conjugation experiments and complete genome sequence using Illumina MiniSeq and Oxford Nanopore sequencing platforms. The two K. pneumoniae isolates were clonal, belonging to ST15 and were detected to carry 19 different antimicrobial resistance genes, including blaNDM-1. Both the isolates carried blaNDM-1 on a self-transmissible IncFII(K):IncR plasmid of 122,804 bp in size with other antimicrobial resistance genes against aminoglycosides (aac(6')-Ib, aadA1, aph(3')-VI), β-lactams (blaCTXM-15, blaOXA-9, blaTEM-1A), fluoroquinolones (aac(6')-Ib-cr), and quinolones (qnrS1). A. baumannii AO22 carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid of 10,880 bp in size. Interestingly, A. baumannii AO22 harbored AbaR4-like genomic resistance island (GI) of 41,665 bp carrying antimicrobial resistance genes against tetracycline [tet(B)], sulfonamides (sul2), and streptomycin (strAB). Here, we identified Japanese carbapenemase-producing Gram-negative bacterias isolated from vegetables posing a food safety issue and a public health concern. Additionally, we reported a GR2-type plasmid carrying two copies of blaOXA-72 and an AbaR4-like resistance island from a foodborne A. baumannii. IMPORTANCE Carbapenemase-producing Gram-negative bacteria (CPGNB) cause severe health care-associated infections and constitute a major public health threat. Here, we investigated the genetic features of CPGNB isolated from fresh vegetable samples in Japan and found CPGNB, including Klebsiella pneumoniae and Acinetobacter baumannii, with dissimilar carbapenemases. The rarely described NDM carbapenemase in Japan was detected in two K. pneumoniae isolates. A. baumannii isolate, identified in this study, carried blaOXA-66 on the chromosome, while blaOXA-72 was found as two copies on a GR2-type plasmid. This study illustrated that even one fresh ready-to-eat vegetable sample might serve as a significant source of resistance genes (blaNDM-1, blaOXA-72, blaCTX-M-14b, and blaCTX-M-15) to frontline and clinically important antibiotics (carbapenems and cephalosporins). Furthermore, the detection of these organisms in fresh vegetables in Japan is alarming and poses a food safety issue and a public health concern.

Acquisition of a genomic resistance island (AbGRI5) from global clone 2 through homologous recombination in a clinical Acinetobacter baumannii isolate

Objectives To reconstruct the evolutionary history of the clinical Acinetobacter baumannii XH1056, which lacks the Oxford scheme allele gdhB. Methods Susceptibility testing was performed using broth microdilution and agar dilution. The whole-genome sequence of XH1056 was determined using the Illumina and Oxford Nanopore platforms. MLST was performed using the Pasteur scheme and the Oxford scheme. Antibiotic resistance genes were identified using ABRicate. Results XH1056 was resistant to all antibiotics tested, apart from colistin, tigecycline and eravacycline. MLST using the Pasteur scheme assigned XH1056 to ST256. However, XH1056 could not be typed with the Oxford MLST scheme as gdhB is not present. Comparative analyses revealed that XH1056 contains a 52 933 bp region acquired from a global clone 2 (GC2) isolate, but is otherwise closely related to the ST23 A. baumannii XH858. The acquired region in XH1056 also contains a 34 932 bp resistance island that resembles AbGRI3 and contains the armA, msrE-mphE, sul1, blaPER-1, aadA1, cmlA1, aadA2, blaCARB-2 and ere(B) resistance genes. Comparison of the XH1056 chromosome to that of GC2 isolate XH859 revealed that the island in XH1056 is in the same chromosomal region as that in XH859. As this island is not in the standard AbGRI3 position, it was named AbGRI5. Conclusions XH1056 is a hybrid isolate generated by the acquisition of a chromosomal segment from a GC2 isolate that contains a resistance island in a new location—AbGRI5. As well as generating ST256, it appears likely that a single recombination event is also responsible for the acquisition of AbGRI5 and its associated antibiotic resistance genes.

Diversity of SCCmec Elements in Methicillin-Resistant Staphylococcus Aureus (MRSA) Recovered from a Healthy Student Population in Kenya

Background: Methicillin-resistant S. aureus continues to be a concern for public health systems, particularly due to infections emerging in non-hospital settings. Resistance to methicillin is presently classified as a serious phenomenon because the majority of methicillin-resistant strains are also multi-drug resistant. The genetic determinant of resistance to methicillin and other β-lactam antibiotics is the mec-A gene, which lies in the SCCmec resistance island. In Kenya, studies done previously have shown the existence of SCCmec types in clinical isolates, but similar information on isolates recovered from healthy populations is scanty.Methods: A cross-sectional study was conducted on healthy university students residing within the university residence halls to determine the carriage of MRSA. MRSA was detected using Cefoxitin (30µg), and mec-A gene and Sccmec elements were detected using conventional PCR methods. A total of 237 students were recruited, and 657 swabs were collected using standards methods for recovering S. aureus.Results: A total of 231 S. aureus isolates were recovered, out of which 26 (11.3%) were MRSA. Out of the 26 MRSA strains, 17 carried the mecA gene in their gene cassettes. SCCmecV was the most prevalent (61.5%), followed by SCCmecII (53.9%) among the MRSA strains. SCCmecIVa, SCCmecIVb, SCCmecIVc and SCCmecIVd were absent in all the isolates. SCCmecV was found to be highly prevalent (64.7%) followed by SCCmecII, 8 (47.1%) among the mecA-positive MRSA strains. On the other hand, small proportions of mecA-negative isolates harbored SCCmecI (0.9%), SCCmecII (3.3%), SCCmec III (0.5%) and SCCmecV (2.3%).Conclusion: This study revealed that the strains recovered from the student population were highly diverse in terms of the SCCmec elements they carried in their gene cassettes.

Scarless Removal of Large Resistance Island AbaR Results in Antibiotic Susceptibility and Increased Natural Transformability in Acinetobacter baumannii

ABSTRACT With a great diversity in gene composition, including multiple putative antibiotic resistance genes, AbaR islands are potential contributors to multidrug resistance in Acinetobacter baumannii. However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE, which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the open reading frame (ORF) of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs, with predicted resistance to several classes of antibiotics, and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable and therefore easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates

Objectives To investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates. Methods Using a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target. Results Genomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/β-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene. Conclusions A novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

Identification of Proteus genomic island 2 variants in two clonal Proteus mirabilis isolates with coexistence of a novel genomic resistance island PmGRI1

Objectives To characterize the MDR genomic islands (GIs) in Proteus mirabilis isolates. Methods Two P. mirabilis strains (C55 and C74) of chicken origin were subjected to WGS (HiSeq and PacBio) and the MDR GIs were determined. Results P. mirabilis strains C55 and C74 are clonal strains and harbour different Proteus genomic island 2 (PGI2) variants (PGI2-C55 and PGI2-C74). The MDR region of PGI2-C55 is composed of two class 1 integrons, separated by a region containing seven copies of IS26 and eight resistance genes, including blaCTX-M-3 and fosA3. The region in PGI2-C74 is a complete In4-type class 1 integron, harbouring five gene cassettes (dfrA16, blaCARB-2, aadA2, cmlA1 and aadA1). In addition, C55 and C74 carry an SXT/R391 integrative and conjugative element (ICEPmiJpn1), harbouring blaCMY-2, and a novel 50.46 kb genomic resistance island named PmGRI1-C55. PmGRI1-C55 harbours a tyrosine-type recombinase/integrase that might be responsible for the integration of PmGRI1-C55 at the 3′ end of tRNA-Sec. It carries an MDR region derived from Tn2670 that harbours a Tn21 region and carries six resistance genes (catA1, blaTEM-1b, aphA1a, sul2, strA and strB). Blast analysis showed diverse PmGRI1 variants in P. mirabilis and Escherichia coli strains. Conclusions The finding of the two new PGI2 variants highlights that the homologous recombination between shared components of class 1 integrons and transposition by IS26 promote the diversity of MDR regions in PGI2. PmGRI1 is a new GI that carries various resistance genes identified in P. mirabilis and E. coli.

Scarless removal of large resistance island AbaR results in antibiotic susceptibility and increased natural transformability in Acinetobacter baumannii

ABSTRACTWith a great diversity in gene composition including multiple putative antibiotic-resistance genes, AbaR islands are potential contributors to multi-drug resistance in Acinetobacter baumannii. However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the ORF of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs with a predicted resistance to several classes of antibiotics and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable, and therefore, easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.