Colistin Resistance in Monophasic Isolates of Salmonella enterica ST34 Collected From Meat-Derived Products in Spain, With or Without CMY-2 Co-production

Colistin is a last-resort antibiotic in fighting severe infections caused by multidrug resistant Gram negative pathogens in hospitals. Zoonotic bacteria acquire colistin resistance in animal reservoirs and mediate its spread along the food chain. This is the case of non-typhoid serovars of Salmonella enterica. Colistin-resistant S. enterica in foods represents a threat to human health. Here, we assessed the prevalence of colistin-resistance in food-borne isolates of S. enterica (2014–2019; Asturias, Spain), and established the genetic basis and transferability of this resistance. Five out of 231 isolates tested (2.2%) were resistant to colistin. Four of them, belonging to the European monophasic ST34 clone of S. Typhimurium, were characterized in the present study. They were collected from pork or pork and beef meat-derived products, either in 2015 (three isolates) or 2019 (one isolate). Molecular typing with XbaI-PFGE and plasmid profiling revealed distinct patterns for each isolate, even though two of the 2015 isolates derived from the same sample. The MICs of colistin ranged from 8 to 16 mg/L. All isolates carried the mcr-1.1 gene located on conjugative plasmids of the incompatibility groups IncX4 (2015 isolates) or IncHI2 (2019 isolate). Apart from colistin resistance, the four isolates carried chromosomal genes conferring resistance to ampicillin, streptomycin, sulfonamides and tetracycline [blaTEM–1, strA-strB, sul2, and tet(B)] and heavy metals, including copper and silver (silESRCFBAGP and pcoGE1ABCDRSE2), arsenic (arsRSD2A2BCA1D1) ± mercury (merEDACPTR), which are characteristically associated with the European ST34 monophasic clone. The 2019 isolate was also resistant to other antibiotics, comprising third generation cephalosporins and cephamycins. The latter phenotype was conferred by the blaCMY–2 gene located on an IncI1-I(α)-ST2 plasmid. Results in the present study identified meat-derived products as a reservoir of a highly successful clone harboring transferable plasmids which confer resistance to colistin and other clinically important antibiotics. An important reduction in the number of food-borne S. enterica detected during the period of the study, together with the low frequency of colistin resistance, underlines the success of One Health initiatives, such as those implemented at the UE, to control zoonotic bacteria along the food chain and to halt the spread of antimicrobial resistance.

ESBL Producing Escherichia coli in Faecal Sludge Treatment Plants: An Invisible Threat to Public Health in Rohingya Camps, Cox's Bazar, Bangladesh

Introduction: Human faecal sludge contains diverse harmful microorganisms, making it hazardous to the environment and public health if it is discharged untreated. Faecal sludge is one of the major sources of E. coli that can produce extended-spectrum β-lactamases (ESBLs).Objective: This study aimed to investigate the prevalence and molecular characterization of ESBL-producing E. coli in faecal sludge samples collected from faecal sludge treatment plants (FSTPs) in Rohingya camps, Bangladesh.Methods: ESBL producing E. coli were screened by cultural as well as molecular methods and further characterized for their major ESBL genes, plasmid profiles, pathotypes, antibiotic resistance patterns, conjugation ability, and genetic similarity.Results: Of 296 isolates, 180 were phenotypically positive for ESBL. All the isolates, except one, contained at least one ESBL gene that was tested (blaCTX−M−1, blaCTX−M−2, blaCTX−M−8, blaCTX−M−9, blaCTX−M−15, blaCTX−M−25, blaTEM, and blaSHV). From plasmid profiling, it was observed that plasmids of 1–211 MDa were found in 84% (151/180) of the isolates. Besides, 13% (24/180) of the isolates possessed diarrhoeagenic virulence genes. From the remaining isolates, around 51% (79/156) harbored at least one virulence gene that is associated with the extraintestinal pathogenicity of E. coli. Moreover, 4% (3/156) of the isolates were detected to be potential extraintestinal pathogenic E. coli (ExPEC) strains. Additionally, all the diarrhoeagenic and ExPEC strains showed resistance to three or more antibiotic groups which indicate their multidrug-resistant potential. ERIC-PCR differentiated these pathogenic isolates into seven clusters. In addition to this, 16 out of 35 tested isolates transferred plasmids of 32–112 MDa to E. coli J53 recipient strain.Conclusion: The present study implies that the faecal sludge samples examined here could be a potential origin for spreading MDR pathogenic ESBL-producing E. coli. The exposure of Rohingya individuals, living in overcrowded camps, to these organisms poses a severe threat to their health.

Molecular Characterisation and Plasmid Profiling of Hydrocarbon Utilizing Bacteria Isolates from Wetlands in Rivers State, Southern Nigeria

Wetlands can intercept runoff from surfaces prior to reaching open water and remove pollutants through physical, chemical, and biological processes thereby protecting and preserving the environment. Because of unsustainable oil exploration activities, most wetlands in Rivers State, Southern Nigeria have suffered severe petroleum-damaged ecosystems. This research was carried out to characterize and identify the hydrocarbon utilizing bacteria associated with crude oil polluted wetlands and to screen for the presence of plasmids that could confer resistance to antibiotics using both cultural and molecular methods. Soil samples were collected from three different wetlands across the state with hand auger at two depths of 0-15cm and 15-30cm twice monthly for three months. The presence of microbial activity was determined by the enumeration and isolation of total heterotrophic and hydrocarbon utilizing bacteria. Eight (8) most occuring hydrocarbon utilizing bacterial isolates were isolated and identified culturally and phenotypically from the 54 wetland soil samples. These bacteria isolates were confirmed to be Bacillus flexus, Bacillus subtilis, Lysinibacillus macroides, Staphylococcus aureus, Chryseobacterium aquifrigidense, Pseudomonas aeruginosa and Salmonella enterica molecularly via sequencing of the 16S rRNA gene. The most common bacteria isolated were Bacillus species, followed by Pseudomonas at a dilution of 106. Seven (7) out of the eight (8) isolates (except Salmoella enterica) showed the presence of the 25kb plasmids at various intensities.

Plasmid profiling of multiple antibiotic-resistant Pseudomonas aeruginosa isolated from soil of the industrial area in Chittagong, Bangladesh

Background Multiple antibiotic-resistant (MAR) Pseudomonas aeruginosa (P. aeruginosa) plays a significant role in triggering nosocomial infection in clinical settings. While P. aeruginosa isolated from the environment is often regarded as non-pathogenic, the progressive development of antibiotic resistance necessitates exploring the MAR patterns and transposable genetic elements like plasmid in the isolates. Results Using ecfX gene-based PCR, 32 P. aeruginosa isolates among 48 soil samples collected from the industrial region have been confirmed. The antibiotic susceptibility pattern of those isolates revealed that 5 (15.63%) of them were resistant to a range of antibiotics, and they were categorized as MAR isolates. Nevertheless, all MAR isolates were found resistant to piperacillin and gentamicin, but none of them to ceftazidime, aztreonam, and ciprofloxacin. Moreover, the isolates were also showed resistance to amikacin (60%), tobramycin (80%), netilmicin (80%), imipenem (60%), doripenem (40%), meropenem (60%), and cefixime (40%). Furthermore, 60% of MAR isolates possessed double plasmids of 1000–2000 bp sizes which indicates the distribution of antibiotic resistance genes in MAR P. aeruginosa might be correlated with the presence of those plasmids. The MAR index’s high threshold values (> 0.20) implied that the isolates were from high-risk environmental sites where the presence of numerous antibiotic residues happened. Conclusions These findings highlighted the presence of multiple antibiotic resistance in P. aeruginosa of the industrial soil and a considerable prospect of transferring antibiotic resistance genes in the microbial community by plasmids. We recommend taking immediate stringent measures to prohibit the unnecessary and overuse of antibiotics in agricultural, industrial, or other purposes.

Anticonjugation and Antibiofilm Evaluation of Probiotic Strains Lactobacillus plantarum 22F, 25F, and Pediococcus acidilactici 72N Against Escherichia coli Harboring mcr-1 Gene

Several species of lactic acid bacteria (LAB) are commonly used as probiotics and as an alternative to antibiotics in various industries, especially in the livestock industry. This study aimed to investigate the anticonjugation and antibiofilm activity of cell-free supernatant (CFS) of Thai LAB strains (Lactobacillus plantarum 22F, 25F, and Pediococcus acidilactici 72N) against colistin-resistant Escherichia coli isolates. A total of six colistin-resistant E. coli strains were isolated from different sources, including pigs, farmers, and farmhouse environments. The E. coli were characterized by plasmid profiling, PCR detection of mcr-1 gene, and antibiotic susceptibility patterns. The CFS at dilutions ≥1:16 was chosen as the proper dilution for anticonjugation assay. Besides, it could significantly reduce the transfer frequencies of resistance gene mcr-1 up to 100 times compared to the neutralizing CFS (pH 6.5). The biofilm production in the planktonic stage was reduced by non-neutralizing and neutralizing CFS determining with crystal violet staining assay up to 82 and 60%, respectively. Moreover, the non-neutralizing CFS also inhibited the biofilm formation in the sessile stage up to 52%. The biofilm illustration was confirmed by scanning electron microscopy (SEM). These results agreed with the findings of the crystal violet technique, which showed a significant reduction in cell density, aggregation, and extracellular polysaccharide (EPS) matrix. The application of Thai LAB may serve as an attractive alternative to antibiotics for reducing biofilm formation and limiting the proliferation of antibiotic-resistant genes.

ESBL and AmpC β-Lactamase Encoding Genes in E. coli From Pig and Pig Farm Workers in Vietnam and Their Association With Mobile Genetic Elements

Animals are considered important sources of ESBL/AmpC-producing bacteria in humans. We analyzed indications of transfer of ESBL/AmpC genes between pigs and pig farmers in Vietnam by analyzing whole genome sequences of 114 ESBL/AmpC-producing E. coli isolated from the two hosts, and performed conjugation experiments and plasmid profiling to confirm that such transfer could have happened. ESBL-encoding genes detected in pigs and pig farmers included blaCTX–M-55, blaCTX-M-27, blaCTX-M-65, blaCTX-M-15, blaCTX-M-14, blaCTX-M-3, blaCTX-M-24, and blaCARB-2, and AmpC β-lactamases included blaCMY-2, blaDHA-1, and blaCMY-42. The most frequent ESBL gene, blaCTX-M-55, was carried on plasmid with replicons types IncF, IncX, IncH, IncN, IncR, and IncP. The insertion transposases downstream of the blaCTX-M-55 gene were different in plasmids carried by different strains. The second most detected gene, blaCTX-M-27, is found in a stable genetic arrangement with the same flanking transposons seen across strains, and the gene was located on similar conjugal IncF plasmid types, suggesting a horizontal spread of these plasmids. In three strains, we observed a novel blaCTX-M-27 harboring IncF type of plasmid which had not been reported before. Its closest reference in NCBI was the non-ESBL Salmonella Typhimurium plasmid pB71 that might have experienced an insertion of blaCTX-M-27. Our data also point to an emergence of plasmids co-carrying ESBL genes, mcr genes, quinolones and other antimicrobials resistance determinants, and such plasmids require special attention. Plasmids phylogeny confirmed that the blaCTX-M-55 encoding plasmids varied considerably, while those encoding blaCTX-M-27 were closely related. Plasmids harboring both ESBL genes were confirmed to be conjugative and not to differ in transfer efficacy. The isolates carrying the plasmids, even those with plasmids of similar types, showed wide genetic variation with high number of SNPs, suggesting horizontal spread of plasmids into different clonal lines. Their virulence profiles did not confirm to known pathotypes, suggesting that unrelated commensals are a main reservoir for ESBL and AmpC β-lactamases in both humans and pigs. Overall, despite evidence of transferability of plasmids in the analyzed strains, our findings do not support that ESBL-producing E. coli from pigs or their ESBL/AmpC encoding plasmids are commonly spread to workers in close contact with the animals.

Clonal relatedness and plasmid profiling of extensively drug-resistant New Delhi metallo-β-lactamase-producing Klebsiella pneumoniae clinical isolates

Aim: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) particularly New Delhi metallo-β-lactamase (NDM) is a serious public health concern globally. The aim of the study to determine the molecular epidemiology of blaNDM-producing clinically isolated K. pneumoniae. Methods: Carbapenem-resistant K. pneumoniae isolates (n = 100) were collected from tertiary care hospital Lahore. Isolates were confirmed by VITEK® 2 system and MALDI-TOF. Minimum inhibitory concentration was performed by VITEK 2 and molecular characterization was done by PCR, PFGE, DNA hybridization and replicon typing. Results: Of 90 MBL-producing K. pneumoniae, 75 were NDM producers; 60 were NDM-1 and 11 NDM-5. A total of 27 K. pneumoniae belonged to ST11 and 14 to ST147. NDM positive isolates were 100% resistant to β-lactam antibiotics except for colistin. 13.3% isolates carried blaNDM on ∼140 kb plasmids. A total of 32 (52.4%) isolates were positive for IncA/C and 18 (29.5%) IncF/II. Conclusion: The extensively resistant lineage of NDM-producing K. pneumoniae is prevalent in the clinical setting.

Antibiotic Resistance and Plasmid Profiling of Escherichia coli Isolated from Human Sewage Samples

In developing countries, the occurrence of antibiotic resistance is increasing day by day and antibiotic resistant microorganisms are being found in almost every environmental setting. Plasmids are considered as the main vector in the procurement and propagation of antibiotic resistance in many microorganisms such as Escherichia coli ( E. coli). The goal of this study was to examine the antibiotic resistance and screening of plasmid in E. coli strains which were previously identified from human sewage samples. During this study antibiotic susceptibility of E. coli isolates were determined by Kirby-Bauer disk diffusion method against 5 antibiotics (ampicilin, ceftriaxone, amoxicillin, ciprofloxacin, azithromycin). Furthermore, plasmid extraction of each isolate was done according to the protocol of FavorPrepTMPlasmid Mini Kit and plasmid profiling was done by agarose gel electrophoresis. In antibiotic sensitivity test, all E. coli strains showed resistance to ampicilin, amoxicillin, and ceftriaxone. In the plasmid profiling, it was revealed that all the isolates of E. coli harbored plasmids. The plasmid sizes ranged from approximately 1.5 to 15 kb. The findings of this study prove the consequences of antibiotic resistance as well as relationship of plasmid with antibiotic resistance which necessitates proper surveillance on antibiotic usage in the developing countries.

Antimicrobial resistance and metallo-beta-lactamase producing among commensal Escherichia coli isolates from healthy children of Khuzestan and Fars provinces; Iran

Background: The emergence of metallo-β-lactamase (MBL)-producing isolates is alarming since they carry mobile genetic elements with great ability to spread; therefore, early detection of these isolates, particularly their reservoir, is crucial to prevent their inter- and intra-care setting dissemination and establish suitable antimicrobial therapies. The current study was designed to evaluate the frequency of antimicrobial resistance (AMR), MBL producers and identification of MBL resistance genes in Escherichia coli strains isolated from fecal samples of the healthy children under three years old. A total of 412 fecal E. coli isolates were collected from October 2017 to December 2018. The study population included healthy infants and children aged <3 years who did not exhibit symptoms of any diseases, especially gastrointestinal diseases. E. coli isolates were assessed to determine the pattern of AMR. E. coli isolates were assessed to determine the pattern of AMR, the production of extended spectrum β-lactamase (ESBL) and MBL by phenotypic methods. Carbapenem-resistant isolates were investigated for the presence of MBL and carbapenemase genes, plasmid profiling, and the ability of conjugation.Results: In sum, AMR, multi-drug resistance (MDR) and ESBL production were observed in more than 54.9%, 36.2% and 11.7% of commensal E. coli isolates, respectively. Out of six isolates resistant to imipenem and meropenem, four isolates were phenotypically detected as MBL producers. Two and one E. coli strains carried the blaNDM-1 and blaVIM-2 genes, respectively and were able to transmit imipenem resistance through conjugation. Conclusion: Our findings showed that children not exposed to antibiotics can be colonized by E. coli isolates resistant to the commonly used antimicrobial compounds and can be a good indicator for the occurrence and prevalence of AMR in the community. These bacteria can act as a potential reservoir of AMR genes including MBL genes of pathogenic bacteria and lead to the dissemination of resistance mechanisms to other bacteria.

Antimicrobial resistance and metallo-beta-lactamase producing among commensal Escherichia coli isolates from healthy children of Khuzestan and Fars provinces; Iran

Background The emergence of metallo-β-lactamase (MBL)-producing isolates is alarming since they carry mobile genetic elements with great ability to spread; therefore, early detection of these isolates, particularly their reservoir, is crucial to prevent their inter- and intra-care setting dissemination and establish suitable antimicrobial therapies. The current study was designed to evaluate the frequency of antimicrobial resistance (AMR), MBL producers and identification of MBL resistance genes in Escherichia coli strains isolated from fecal samples of the healthy children under 3 years old. A total of 412 fecal E. coli isolates were collected from October 2017 to December 2018. The study population included healthy infants and children aged < 3 years who did not exhibit symptoms of any diseases, especially gastrointestinal diseases. E. coli isolates were assessed to determine the pattern of AMR. E. coli isolates were assessed to determine the pattern of AMR, the production of extended spectrum β-lactamase (ESBL) and MBL by phenotypic methods. Carbapenem-resistant isolates were investigated for the presence of MBL and carbapenemase genes, plasmid profiling, and the ability of conjugation. Results In sum, AMR, multi-drug resistance (MDR) and ESBL production were observed in more than 54.9, 36.2 and 11.7% of commensal E. coli isolates, respectively. Out of six isolates resistant to imipenem and meropenem, four isolates were phenotypically detected as MBL producers. Two and one E. coli strains carried the blaNDM-1 and blaVIM-2 genes, respectively and were able to transmit imipenem resistance through conjugation. Conclusion Our findings showed that children not exposed to antibiotics can be colonized by E. coli isolates resistant to the commonly used antimicrobial compounds and can be a good indicator for the occurrence and prevalence of AMR in the community. These bacteria can act as a potential reservoir of AMR genes including MBL genes of pathogenic bacteria and lead to the dissemination of resistance mechanisms to other bacteria.