Carriage of antibiotic resistant bacteria in endangered and declining Australian pinniped pups

The rapid emergence of antimicrobial resistance (AMR) is a major concern for wildlife and ecosystem health globally. Genetic determinants of AMR have become indicators of anthropogenic pollution due to their greater association with humans and rarer presence in environments less affected by humans. The objective of this study was to determine the distribution and frequency of the class 1 integron, a genetic determinant of AMR, in both the faecal microbiome and in Escherichia coli isolated from neonates of three pinniped species. Australian sea lion ( Neophoca cinerea ), Australian fur seal ( Arctocephalus pusillus doriferus ) and long-nosed fur seal ( Arctocephalus forsteri ) pups from eight breeding colonies along the Southern Australian coast were sampled between 2016-2019. DNA from faecal samples ( n =309) and from E. coli ( n =795) isolated from 884 faecal samples were analysed for class 1 integrons using PCRs targeting the conserved integrase gene ( intI ) and the gene cassette array. Class 1 integrons were detected in A. p. doriferus and N. cinerea pups sampled at seven of the eight breeding colonies investigated in 4.85% of faecal samples ( n =15) and 4.52% of E. coli isolates ( n =36). Integrons were not detected in any A. forsteri samples. DNA sequencing of the class 1 integron gene cassette array identified diverse genes conferring resistance to four antibiotic classes. The relationship between class 1 integron carriage and the concentration of five trace elements and heavy metals was also investigated, finding no significant association. The results of this study add to the growing evidence of the extent to which antimicrobial resistant bacteria are polluting the marine environment. As AMR determinants are frequently associated with bacterial pathogens, their occurrence suggests that these pinniped species are vulnerable to potential health risks. The implications for individual and population health as a consequence of AMR carriage is a critical component of ongoing health investigations.

First Report of aacC5-aadA7Δ4 Gene Cassette Array and Phage Tail Tape Measure Protein on Class 1 Integrons of Campylobacter Species Isolated from Animal and Human Sources in Egypt

Campylobacter species are common commensals in the gastrointestinal tract of livestock animals; thus, animal-to-human transmission occurs frequently. We investigated for the first time, class 1 integrons and associated gene cassettes among pan drug-resistant (PDR), extensively drug-resistant (XDR), and multidrug-resistant (MDR) Campylobacter species isolated from livestock animals and humans in Egypt. Campylobacter species were detected in 58.11% of the analyzed chicken samples represented as 67.53% Campylobacter jejuni(C. jejuni) and 32.47% Campylobacter coli (C. coli). C. jejuni isolates were reported in 51.42%, 74.28%, and 66.67% of examined minced meat, raw milk, and human stool samples, respectively. Variable antimicrobial resistance phenotypes; PDR (2.55%), XDR (68.94%), and MDR (28.5%) campylobacters were reported. Molecular analysis revealed that 97.36% of examined campylobacters were integrase gene-positive; all harbored the class 1 integrons, except one possessed an empty integron structure. DNA sequence analysis revealed the predominance of aadA (81.08%) and dfrA (67.56%) alleles accounting for resistance to aminoglycosides and trimethoprim, respectively. This is the first report of aacC5-aadA7Δ4 gene cassette array and a putative phage tail tape measure protein on class 1 integrons of Campylobacter isolates. Evidence from this study showed the possibility of Campylobacter–bacteriophage interactions and treatment failure in animals and humans due to horizontal gene transfer mediated by class 1 integrons.

Molecular Analysis of a blaIMP-1-Harboring Class 3 Integron in Multidrug-Resistant Pseudomonas fulva

ABSTRACT A multidrug-resistant (MDR) Pseudomonas fulva strain was isolated in 2006 from a urine sample. The isolate harbored the blaIMP-1 gene, which was located in a chromosomal Tn402-like class 3 integron as a gene cassette array of aacA31-fosE-blaIMP-1. Two mutations in gyrA and one mutation in parC were detected in quinolone-resistance-determining regions (QRDRs). We report a full-length, novel, blaIMP-1-carrying class 3 integron. This integron, together with mutations in QRDRs, could have influenced the MDR phenotype.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (Pc), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position impacts the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

AbaR5, a Large Multiple-Antibiotic Resistance Region Found in Acinetobacter baumannii

ABSTRACT A multiply antibiotic-resistant Acinetobacter baumannii strain, 3208, contains the aacC1-orfP-orfP-orfQ-aadA1 gene cassette array; sul1, tetA(A), and aphA1b genes; and a mer operon in a large region containing a novel transposon, Tn6020, and segments of Tn1696, Tn21, Tn1721, and Tn5393. This region is part of a genomic resistance island, AbaR5, related to and found in the same chromosomal position as AbaR1. This strain is the first European clone I isolate detected in Australia.

Dissemination of sul3-Containing Elements Linked to Class 1 Integrons with an Unusual 3′ Conserved Sequence Region among Salmonella Isolates

ABSTRACT A sul3 domain (IS440-sul3-orf1-IS26) was found linked to an unusual 3′ conserved sequence region (qacH) of class 1 integrons and detected among nontyphoid Salmonella isolates (n = 47) from different sources. Three types of integrons differing in the gene cassette array (dfrA12-orfF-aadA2-cmlA1-aadA1, dfrA12-orfF-aadA2/1, and estX-psp-aadA2-cmlA1-aadA1) were found associated with this sul3 domain. They were associated with particular clones and specific high-molecular-weight plasmids.

In117, an Unusual In0-Like Class 1 Integron Containing CR1 and blaCTX-M-2 and Associated with a Tn21-Like Element

ABSTRACT An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and bla CTX-M-2 was characterized from Escherichia coli. The integron contained an unusual gene cassette array, estX-aadA1, embedded between the 5′-conserved segment (5′-CS) and 3′-CS1 regions and was flanked by mer-Tn21 sequences downstream of the tni truncated module. This element constitutes one of the few examples of CR1-bearing class 1 integrons that has been fully characterized.