Pseudomonas guariconensis Necrotizing Fasciitis, United Kingdom.

We describe a case of necrotizing fasciitis in the United Kingdom in which Pseudomonas guariconensis was isolated from multiple blood culture and tissue samples. The organism carried a Verona integron-encoded metallo-ß-lactamase gene and evidence of decreased susceptibility to ß-lactam antimicrobial agents. Clinicians should use caution when treating infection caused by this rare pathogen.

Quantitative microbial risk assessment for ingestion of antibiotic resistance genes from private wells contaminated by human and livestock fecal sources.

We used quantitative microbial risk assessment to estimate ingestion risk for intI1, erm(B), sul1, tet(A), tet(W), and tet(X) in private wells contaminated by human and/or livestock feces. Genes were quantified with five human-specific and six bovine-specific microbial source-tracking (MST) markers in 138 well-water samples from a rural Wisconsin county. Daily ingestion risk (probability of swallowing ≥1 gene) was based on daily water consumption and a Poisson exposure model. Calculations were stratified by MST source and soil depth over the aquifer where wells were drilled. Relative ingestion risk was estimated using wells with no MST detections and >6.1 m soil depth as a referent category. Daily ingestion risk varied from 0 to 8.8 × 10-1 by gene and fecal source (i.e., human or bovine). The estimated number of residents ingesting target genes from private wells varied from 910 (tet(A)) to 1,500 (intI1 and tet(X)) per day out of 12,000 total. Relative risk of tet(A) ingestion was significantly higher in wells with MST markers detected, including wells with ≤6.1 m soil depth contaminated by bovine markers (2.2 [90% CI: 1.1-4.7]), wells with >6.1 m soil depth contaminated by bovine markers (1.8 [1.002-3.9]), and wells with ≤6.1 m soil depth contaminated by bovine and human markers simultaneously (3.1 [1.7-6.5]). Antibiotic resistance genes (ARGs) were not necessarily present in viable microorganisms, and ingestion is not directly associated with infection. However, results illustrate relative contributions of human and livestock fecal sources to ARG exposure and highlight rural groundwater as a significant point of exposure.IMPORTANCEAntibiotic resistance is a global public health challenge with well-known environmental dimensions, but quantitative analyses of the roles played by various natural environments in transmission of antibiotic resistance are lacking, particularly for drinking water. This study assesses risk of ingestion for several antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in drinking water from private wells in a rural area of northeast Wisconsin, United States. Results allow comparison of drinking water as an exposure route for antibiotic resistance relative to other routes like food and recreational water. They also enable a comparison of the importance of human versus livestock fecal sources in the study area. Our study demonstrates the previously unrecognized importance of untreated rural drinking water as an exposure route for antibiotic resistance and identifies bovine fecal material as an important exposure factor in the study setting.

Comparison of integron mediated antimicrobial resistance in clinical isolates of Escherichia coli from urinary and bacteremic sources.

BACKGROUND: Antimicrobial resistance (AMR) is a global threat driven mainly by horizontal gene transfer (HGT) mechanisms through mobile genetic elements (MGEs) including integrons. The variable region (VR) of an integron can acquire or excise gene cassettes (GCs) that confer resistance to antibiotics based on the selection pressure. Escherichia coli plays a significant role in the genetic transfer of resistance determinants to other Gram-negative bacteria. Current study is aimed to detect and compare integron-mediated resistance in clinical isolates of E. coli. Unique isolates of E. coli from urine or blood cultures were studied for their antimicrobial resistance patterns and integrons were detected using polymerase chain reaction assays followed by Sanger sequencing of GCs. RESULTS: During the study period, a total of 470 E. coli isolates were obtained, 361 (76.8%) from urinary and 109 (23.1%) from bacteremic sources. Class 1 integrons were detected in 66 (18.2%) and 26 (23.8%) isolates respectively. Urinary isolates of E. coli harbouring Class 1 integrons demonstrated significantly higher rates of resistance (p < 0.05) for most antibiotics (12/16, 75%) compared to integron negative isolates. Although not statistically significant, similar differences were observed in bacteremic isolates. Among the urinary isolates, 27 (40.9%) had a VR, in which the most common GC array detected was DfrA17-AadA5 (n = 14), followed by DfrA5 (n = 4) and DfrA12 (n = 3). Among bacteremic isolates, only 4 (15.3%) had a VR, all of which were carrying DfrA17. The detected GC array correlated with the respective isolates' phenotypic resistance patterns. CONCLUSION: We found a strong correlation between integron positivity and trimethoprim resistance among E. coli from urinary sources. Although higher rates of resistance were observed in bacteremic isolates, they mostly carried empty integrons.

Prevalence and characterization of quinolone resistance and integrons in clinical Gram-negative isolates from Gaza strip, Palestine.

BACKGROUND: Gram-negative bacteria with quinolone resistance and extended-spectrum beta-lactamases (ESBLs) present significant treatment challenges. This study evaluated the prevalence and characteristics of quinolone resistance in Gram-negative strains, investigating the relationship between plasmid-mediated quinolone resistance (PMQR), ESBLs, and integrons. METHODS AND RESULTS: We collected 146 Gram-negative isolates from patients in three Palestinian hospitals. For quinolone resistance isolates, the presence and characterization of PMQR, ß-lactamase genes and integrons were studied by PCR and sequencing. Out of 146 clinical isolates, 64 (43.8%) were resistant to quinolones, with 62 (97%) being multidrug-resistant (MDR) and 33 (51.5%) ESBL-producers. PMQR-encoding genes were present in 45 (70.3%) isolates, including aac(6')-Ib-cr (26.6%), qnrA (18.8%), qnrS1 (20.8%), and qnrB (6.4%). BlaCTX-M genes were detected in 50% (32/64) of isolates, with blaCTX-M-15 being the most common. BlaTEM-1, blaSHV-1 and blaVIM genes were found in 13, 6, and 4 isolates, respectively. Class I integrons were found in 31/64 (48%) of isolates, with 14 containing gene cassettes conferring resistance to trimethoprim (dhfr17, dfrA12, dfrA1) and aminoglycosides resistance genes (aadA1, aadA2, aadA5, and aadA6). CONCLUSIONS: This study found a high rate of quinolone resistance, ESBL and integrons in clinical Gram-negative isolates from our hospitals. Urgent measures are crucial, including implementing an antimicrobial resistance surveillance system, to control and continuously monitor the development of antimicrobial resistance.

Emergence of extensive drug resistance and high prevalence of multidrug resistance among clinical Proteus mirabilis isolates in Egypt.

BACKGROUND: Proteus mirabilis is an opportunistic pathogen that has been held responsible for numerous nosocomial and community-acquired infections which are difficult to be controlled because of its diverse antimicrobial resistance mechanisms. METHODS: Antimicrobial susceptibility patterns of P. mirabilis isolates collected from different clinical sources in Mansoura University Hospitals, Egypt was determined. Moreover, the underlying resistance mechanisms and genetic relatedness between isolates were investigated. RESULTS: Antimicrobial susceptibility testing indicated elevated levels of resistance to different classes of antimicrobials among the tested P. mirabilis clinical isolates (n = 66). ERIC-PCR showed great diversity among the tested isolates. Six isolates (9.1%) were XDR while all the remaining isolates were MDR. ESBLs and AmpCs were detected in 57.6% and 21.2% of the isolates, respectively, where blaTEM, blaSHV, blaCTX-M, blaCIT-M and blaAmpC were detected. Carbapenemases and MBLs were detected in 10.6 and 9.1% of the isolates, respectively, where blaOXA-48 and blaNDM-1 genes were detected. Quinolone resistant isolates (75.8%) harbored acc(6')-Ib-cr, qnrD, qnrA, and qnrS genes. Resistance to aminoglycosides, trimethoprim-sulfamethoxazole and chloramphenicol exceeded 80%. Fosfomycin was the most active drug against the tested isolates as only 22.7% were resistant. Class I or II integrons were detected in 86.4% of the isolates. Among class I integron positive isolates, four different gene cassette arrays (dfrA17- aadA5, aadB-aadA2, aadA2-lnuF, and dfrA14-arr-3-blaOXA-10-aadA15) and two gene cassettes (dfrA7 and aadA1) were detected. While class II integron positive isolates carried four different gene cassette arrays (dfrA1-sat1-aadA1, estXVr-sat2-aadA1, lnuF- dfrA1-aadA1, and dfrA1-sat2). CONCLUSION: P. Mirabilis ability to acquire resistance determinants via integrons may be held responsible for the elevated rates of antimicrobial resistance and emergence of XDR or even PDR strains limiting the available therapeutic options for management of infections caused by those strains.

Prevalence and molecular characterization of multi-resistant Escherichia coli isolates from clinical bovine mastitis in China.

Different antibiotics are used to treat mastitis in dairy cows that is caused by Escherichia coli (E. coli). Antimicrobial resistance in food-producing animals in China has been monitored since 2000. Surveillance data have shown that the prevalence of multiresistant E. coli in animals has increased significantly. This study aimed to investigate the occurrence and molecular characteristics of resistance determinants in E. coli strains (n = 105) obtained from lactating cows with clinical bovine mastitis (CBM) in China. A total of 220 cows with clinical mastitis, which has swollen mammary udder with reduced and red or gangrenous milk, were selected from 5000 cows. The results showed 94.3% of the isolates were recognized as multidrug resistant. The isolates (30.5%) were positive for the class I integrase gene along with seven gene cassettes that were accountable for resistance to trimethoprim resistance (dfrA17, dfr2d and dfrA1), aminoglycosides resistance (aadA1 and aadA5) and chloramphenicol resistance (catB3 and catB2), respectively. The blaTEM gene was present in all the isolates, and these carried the blaCTX gene. A double mutation in gyrA (i.e., Ser83Leu and Asp87Asn) was observed in all fluoroquinolone-resistant isolates. In total, nine fluoroquinolone-resistant E. coli isolates were identified with five different types of mutations in parC. In four (44.4%) isolates, Ser458Ala was present in parE, and in all nine (9/9) fluoroquinolone-resistant isolates, Pro385Ala was present in gyrB. Meanwhile, fluoroquinolone was observed as highly resistant, especially in isolates with gyrA and parC mutations. In summary, the findings of this research recognize the fluoroquinolone resistance mechanism and disclose integron prevalence and ESBLs in E. coli isolates from lactating cattle with CBM.

The swine waste resistome: Spreading and transfer of antibiotic resistance genes in Escherichia coli strains and the associated microbial communities.

The overuse of antimicrobials in livestock farming has led to the development of resistant bacteria and the spread of antibiotic-resistant genes (ARGs) among animals. When manure containing these antibiotics is applied to agricultural fields, it creates a selective pressure that promotes the acquisition of ARGs by bacteria, primarily through horizontal gene transfer. Most research on ARGs focuses on their role in clinical antibiotic resistance and their transfer from environmental sources to bacteria associated with humans, such as Escherichia coli. The study investigates the spread of antibiotic-resistant genes (ARGs) through class 1 integrons in 27 Escherichia coli strains from pig manure. It focuses on six common ARGs (ermB, cmlA, floR, qnrS, tetA, and TEM) and the class 1 integron gene, assessing their prevalence in manure samples from three pig farms. The study found correlations and anticorrelations among these genes, indicating a predisposition of the integron in spreading certain ARGs. Specifically, cmlA and tetA genes were positively correlated with each other and negatively with int1, suggesting they are not transferred via Int1. Farm B had the highest int1 counts and a higher abundance of the TEM gene, but lower levels of cmlA and tetA genes. The results underscore the complexity of predicting ARG spread in agricultural environments and the associated health risks to humans through the food chain. The study's results offer valuable insights into the antibiotic-resistant genes (ARGs) profile in swine livestock, potentially aiding in the development of methods to trace ARGs in the environment.

Unexpected role of pig nostrils in the clonal and plasmidic dissemination of extended-spectrum beta-lactamase-producing Escherichia coli at farm level.

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

Unveiling the ecological landscape of bacterial ß-lactam resistance in Delhi-national capital region, India: An emerging health concern.

Inappropriate antibiotic use not only amplifies the threat of antimicrobial resistance (AMR), moreover exacerbates the spread of resistant bacterial strains and genes in the environment, underscoring the critical need for effective research and interventions. Our aim is to assess the prevalence and resistance characteristics of ß-lactam resistant bacteria (BLRB) and ß-lactamase resistant bacterial genes (BLRBGs) under various environmental conditions within Delhi NCR, India. Using a culture-dependent method, we isolated 130 BLRB from 75 different environmental samples, including lakes, ponds, the Yamuna River, agricultural soil, aquatic weeds, drains, dumping yards, STPs, and gaushalas. Tests for antibiotic susceptibility were conducted in addition to phenotypic and genotypic identification of BLs and integron genes. The water and sediment samples recorded an average bacterial abundance of 3.6 × 106 CFU/mL and an average ampicillin-resistant bacterial count of 2.2 × 106 CFU/mL, which can be considered a potent reservoir of BLRB and BLRBGs. The majority of the BLRB discovered are opportunistic pathogens from the Bacillus, Aeromonas, Pseudomonas, Enterobacter, Escherichia, and Klebsiella genera, with Multiple Antibiotic Resistance (MAR) index ≥0.2 against a wide variety of ß-lactams and ß-lactamase (BLs) inhibitor combinations. The antibiotic resistance pattern was similar in the case of bacteria isolated from STPs. Meanwhile, bacteria isolated from other sources were diverse in their antibiotic resistance profile. Interestingly, we discovered that 10 isolates of various origins produce both Extended Spectrum BLs and Metallo BLs, as well as found harboring blaTEM, blaCTX, blaOXA, blaSHV, int-1, and int-3 genes. Enterobacter cloacae (S50/A), a common nosocomial pathogen isolated from Yamuna River sediment samples at Nizamuddin point, possesses three BLRBGs (blaTEM, blaCTX, and blaOXA) and a MAR index of 1.0, which is a major cause for concern. Therefore, identifying the source, origin and dissemination of BLRB and BLRGs in the environment is of the utmost importance for designing effective mitigation approaches to reduce a load of antimicrobial resistance factors in the environmental settings.

Occurrence, antibiotic resistance and molecular characterisation of Shiga toxin-producing Escherichia coli isolated from broiler chickens in Tunisia.

1. Shiga toxin-producing Escherichia coli (STEC) strains are associated with disease outbreaks which cause a public health problem. The aim of this study was to determine the frequency of STEC strains, their virulence factors, phylogenetic groups and antimicrobial resistance profiles in broiler chickens.2. A total of 222 E.coli isolates were collected from the caecum of chickens intended to be slaughtered. Antibiotic susceptibility was tested against 21 antimicrobial agents and ESBL phenotype was assessed by double-disk synergy test. The presence of STEC virulence genes stx1, stx2,eaeA and ehxA was detected by PCR. The identification of STEC serogroups was realised by PCR amplification. Additive virulence genes, phylogenetic groups and integrons were examined among the STEC isolates.3. Out of 222 E.coli isolates, 72 (32%) were identified as STEC strains and the most predominant serogroups were O103, O145 and O157. Shiga toxin gene 1 (stx1) was found in 84.7% (61/72) of the STEC strains, and eae and stx2 were detected in 38.8% and 13.8%, respectively. The ESBL phenotype was documented in 48.6% (35/72) of isolates. Most of the isolates (90.3%) carried class 1 integron with the gene cassette encoding resistance to trimethoprim (dfrA) and streptomycin (aadA) in 31.9% of the isolates. Class 2 integron was identified in 36.1% of isolates.4. Broilers can be considered as a reservoir of STEC strains which have high virulence factors and integrons that might be transmitted to other chickens, environments and humans. It is important to undertake surveillance and efficient control measures in slaughterhouses and farms to control measures of STEC bacteria.

Integrons and multidrug resistance across phylogenetic groups of clinical isolates of Escherichia coli.

Objective: This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings. Methods: A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays. Results: Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-ß-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively. Conclusion: Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against ß-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.

Pangenome analysis of Corynebacterium striatum: insights into a neglected multidrug-resistant pathogen.

BACKGROUND: Over the past two decades, Corynebacterium striatum has been increasingly isolated from clinical cultures with most isolates showing increased antimicrobial resistance (AMR) to last resort agents. Advances in the field of pan genomics would facilitate the understanding of the clinical significance of such bacterial species previously thought to be among commensals paving the way for identifying new drug targets and control strategies. METHODS: We constructed a pan-genome using 310 genome sequences of C. striatum. Pan-genome analysis was performed using three tools including Roary, PIRATE, and PEPPAN. AMR genes and virulence factors have been studied in relation to core genome phylogeny. Genomic Islands (GIs), Integrons, and Prophage regions have been explored in detail. RESULTS: The pan-genome ranges between a total of 5253-5857 genes with 2070 - 1899 core gene clusters. Some antimicrobial resistance genes have been identified in the core genome portion, but most of them were located in the dispensable genome. In addition, some well-known virulence factors described in pathogenic Corynebacterium species were located in the dispensable genome. A total of 115 phage species have been identified with only 44 intact prophage regions. CONCLUSION: This study presents a detailed comparative pangenome report of C. striatum. The species show a very slowly growing pangenome with relatively high number of genes in the core genome contributing to lower genomic variation. Prophage elements carrying AMR and virulence elements appear to be infrequent in the species. GIs appear to offer a prominent role in mobilizing antibiotic resistance genes in the species and integrons occur at a frequency of 50% in the species. Control strategies should be directed against virulence and resistance determinants carried on the core genome and those frequently occurring in the accessory genome.

Uncovering Bacterial Hosts of Class 1 Integrons in an Urban Coastal Aquatic Environment with a Single-Cell Fusion-Polymerase Chain Reaction Technology.

Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbor class 1 integrons. We developed a modified version of epicPCR (emulsion, paired isolation, and concatenation polymerase chain reaction (PCR)) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first application of epicPCR for targeting variable, multigene loci of interest. We also identified the Rhizobacter genus as novel hosts of class 1 integrons. These findings establish epicPCR as a powerful tool for linking taxa to class 1 integrons in environmental bacterial communities and offer the potential to direct mitigation efforts toward hotspots of class 1 integron-mediated dissemination of AMR.

Integrons as the potential targets for combating multidrug resistance in Enterobacteriaceae using CRISPR- Cas9 technique.

The emergence of multi-drug resistance (MDR) to pan-drug resistance (PDR) in Enterobacteriaceae has made treatment extremely challenging. Genetic mutations and horizontal gene transfer (HGT) through mobile genetic elements (MGEs) were frequently associated mechanisms of drug resistance in pathogens. However, transposons, plasmids, and integrons transfer MDR genes in bacterium via HGT much faster. Integrons are dsDNA segment that plays a crucial role in the adaptation and evolution of bacteria. They contain multiple gene cassettes that code for antibiotic resistance determinants that are expressed by a single promoter (Pc). Integrons are the cause of drug resistance in Enterobacteriaceae. Although alternatives to antibiotics such as bacteriophages, phage proteins, antimicrobial peptides, and natural compounds have been widely used to treat MDR infections, there have been limited efforts to reverse the antibiotic resistance ability of bacteria. Thus, silencing the genes harboured on MGEs achieved by Gene Editing Techniques (GETs) might prevent the spread of MDR. One such GETs, which has a simple design, good repeatability, low cost, and high efficiency, is CRISPR- Cas9 system. Thus, this review is a first of the kind that focuses on utilizing the structure of an integron to make it an ideal target for GETs like CRISPR- Cas9 systems.

Novel aadA5 and dfrA17 variants of class 1 integron in multidrug-resistant Escherichia coli causing bovine mastitis.

Mobile genetic elements (MGEs) are associated with the emergence of multidrug resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. This study explores the role of class 1 integrons and IS26 elements in breaching taxonomic barriers. A total of 110 E. coli bacteria were isolated from 300 clinical mastitis milk samples. The 98% E. coli isolates were extended-spectrum beta-lactamase- producers. About 83% of these isolates carried co-resistance for fluoroquinolones. The co-existence of (extended-spectrum beta-lactamase + quinolone resistance determining region mutations) and (extended-spectrum beta-lactamase + plasmid-mediated quinolone resistance genes) was found in 76% and 44% of isolates, respectively. The MGEs were detected in 88% of isolates with IS26 in 82% and class 1 integrase in 40% of isolates. The types of class 1 integron gene cassettes detected includes dfrA7, (dfrA17 + aadA5), and (dfrA1 + aadA1). We discovered 2 and 4 novel variants of the dfrA17 and aadA5 genes, respectively. We report a variant of aadA5 with mutation E235G in the Indian subcontinent earlier reported only in a human clinical isolate from Belgium. About 19 isolates carried IS26 linked to integrase gene intI1 with an internal deletion of 265 bp in the 5`CS of integrase gene intI1, earlier reported only in E. coli ST131 isolates from human clinical, wastewater samples. This study suggests intercontinental dissemination of antibiotic resistant genes (ARGs) across different microbiomes via mobile genetic elements. KEY POINTS: • The role of mobile genetic elements in the emergence of multidrug-resistant E. coli in bovine mastitis. • Novel variants of the aadA5 (aminoglycoside adenyl transferase) and dfrA17 (dihydrofolate reductase) genes were identified in pathogenic E. coli isolated from bovine mastitis in class 1 integron gene cassette. • Sequence analysis of mobile genetic components revealed the physical connection between IS26 and intI1 genes with an internal deletion in 5'CS of class 1 integrase.

Antibiotic resistance profile and molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) from human clinical samples in gaza strip, palestine.

The rates of antibiotic resistance in extraintestinal pathogenic Escherichia coli (ExPEC) have increased significantly in recent years. This study aims at studying antibiotic resistance, virulence factors (VFs), and the phylogenetic background of ExPEC isolated from Palestinian patients. A total of 42 ExPEC isolates were collected from patients with extraintestinal infections in three Palestinian hospitals. Antimicrobial susceptibility was studied by the disk diffusion method. Resistance/virulence-gene profiles, phylogenetic groups, and integron profiles of these isolates were studied by PCR. The susceptibility to carbapenems was detected in 90.5% of the isolates. Half of the isolates were resistant to ampicillin and sulfamethoxazole/trimethoprim, and 33.3% of the isolates were multidrug-resistant. BlaTEM-1 was detected in seven isolates and blaOXA-1 was identified in one isolate. Sul, qnrA, and aac(6')-Ib-cr genes were found in 12, 3, and 2 isolates, respectively. Class 1 integron has been identified in 10 isolates with the gene cassette arrangement dhfr17 + aadA5. The isolates were distributed between phylogroups B2 and D. The presence of VFs, antibiotic resistance genes, and class 1 integron associated with phylogenetic groups (B2 and D) among multidrug-resistant (MDR)-ExPEC recovered from urinary tract infections (UTIs) patients will complicate infection management and increase therapy failure. Routine screening of antibiotic resistance is important for appropriate and efficient empirical treatment.

Class 1 and 2 integrons and antibiotic resistance profile in Salmonella spp. from San Cristobal River, Laguna, Philippines.

Infection with multidrug resistant bacteria is a significant public health concern. Bacteria culture of water samples (n=120) collected in San Cristobal River, Philippines, showed that half (n=60) were positive for Salmonella spp. Screening of all isolates (n=179) for susceptibility to antibiotics showed that most (76.4%; n=113) were positive for class 1 integrons, of which one isolate was also positive for the class 2 integron. The presence of class 1 integrons was associated with resistance to antibiotics (p<0.05). Sequencing of class 1 integron variable regions (VRs) differeciated 11 gene cassettes: dfrA1 or dfrA17; aadA1 or aadA2; blaCTX-M-2 or bla-OXA-1; SmdAB; CmlA1 and aaC 3-Id. However, sequencing of class 2 integron VR differenciated estX, sat2, and aadA1. These results provide insights into evolutionary changes within bacterial multidrug resistant cassettes, more accurately to estimate heath risk associated with the river water. .

Antimicrobial Resistance and Molecular Characterization of Gene Cassettes from class 1 Integrons in Carbapenem-resistant Escherichia coli strains.

OBJECTIVE: To investigate the distribution of class 1 integrons and their variable regional molecular characteristics, as well as the diversity of promoter and drug sensitivity of CR-Eco (carbapenem-resistant E. coli) strains. METHOD: A total of 117 CR-Eco strains, collected between 2012.01 and 2019.12, underwent fully automated bacterial identification and sensitization using VITEK-2 Compact and supplemented by K-B assay. PCR was employed to screen for class 1 integrase genes and integron variable regions, while the promoter type and variable region gene cassette characteristics were determined by sequencing analysis. RESULTS: The positive rate of the class 1 integron of the CR-Eco strains was 83.70% (92/117) herein. Moreover, class 1 integrase-positive strains exhibited statistically significant resistance to aztreonam, ceftazidime, ciprofloxacin, ceftriaxone, gentamicin, meropenem, and trimethoprim-sulfamethoxazole compared to integron-negative strains (P < 0.05). Variable regions were observed in 77 of the 92 class 1 integrase-positive strains. In addition, seven gene cassettes were detected, namely dfrA17-aadA5, aadA22, dfrA12-aadA2, dfrA12, dfrA17, dfrA27 and aadA. Finally, five types of class 1 integron variable region promoters were identified in those 77 strains, including PcW, PcH1, PcWTGN-10, PcH1TGN-10, and P2, which were detected in 48, 18, 8, 2, and 1 strains, respectively. CONCLUSION: The primary integrator variable region gene cassettes of this class were dfrA and aadA. The integron-positive strains displayed simultaneous high resistance to multiple antimicrobial drugs. The integrator variable region promoters of the CR-Eco strains are primarily weak and can potentially form and spread drug resistance.

Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia.

Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::blaCTX-M-2) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.

Spread of antimicrobial resistance genes via pig manure from organic and conventional farms in the presence or absence of antibiotic use.

AIMS: Antibiotic-resistant bacteria affect human and animal health. Hence, their environmental spread represents a potential hazard for mankind. Livestock farming is suspected to be a key factor for spreading antibiotic resistance; consumers expect organic farming to imply less environmental health risk. This study aimed to assess the role of manure from organic and conventional farms for spreading antimicrobial resistance (AMR) genes. METHODS AND RESULTS: AMR-genes-namely tet(A), tet(B), tet(M), sul2 and qacE/qacEΔ1 (potentially associated with multiresistance) were quantified by qPCR. Antimicrobial use during the study period was qualitatively assessed from official records in a binary mode (yes/no). Median concentrations were between 6.44 log copy-equivalents/g for tet(A) and 7.85 for tet(M) in organic liquid manure, and between 7.48 for tet(A) and 8.3 for sul2 in organic farmyard manure. In conventional manure, median concentrations were 6.67 log copy-equivalents/g for sul2, 6.89 for tet(A), 6.77 for tet(B) and 8.36 for tet(M). Integron-associated qac-genes reached median concentrations of 7.06 log copy-equivalents/g in organic liquid manure, 7.13 in conventional manure and 8.18 in organic farmyard manure. The use of tetracyclines or sulfonamides increased concentrations of tet(A) and tet(M), or of sul2, respectively. Comparing farms that did not apply tetracyclines during the study, the relative abundance of tet(A) and tet(M) was still higher for conventional piggeries than for organic ones. CONCLUSIONS: Relative abundances of AMR genes were higher in conventional farms, compared to organic ones. Antibiotic use was linked to the relative abundance of AMR-genes. However, due to the bacterial load, absolute concentrations of AMR-genes were comparable between fertilizers of organic and conventional farms. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first absolute quantification of AMR-genes in manure from organic farms. Our study underlines the importance of long-term reduction in the use of antimicrobial agents in order to minimize antibiotic resistance.