Antibiotic resistance of E. coli isolates from different water sources in Mbarara, Uganda.

Escherichia coli is widely used as an indicator of recent faecal pollution of water. Most E. coli strains are commensals; however, isolates in water samples have been shown to carry antibiotic resistance determinants. In total, 47 E. coli were isolated from selected drinking water sources in Mbarara, Uganda. The isolates were examined for their susceptibility to seven antibiotics and the presence of nine antibiotic-resistance genes (mostly ß-lactamase genes) and class 1 integrons. Isolates showed a high resistance to ampicillin of 55.5% and a high sensitivity to azithromycin and gentamicin at 98 and 96%, respectively. PCR analysis showed the presence of extended-spectrum ß-lactamase genes blaCTX-M-32 and blaCMY-2 in 64 and 36% of the isolates. The carbapenemase genes blaOXA-48, blaVIM-2, blaNDM-1, and blaKPC-3 were either not detected or only in a very small number of the isolates, whereas class 1 integrons were present in 68% of the isolates. This study proves that antimicrobial resistance exists in E. coli in water used for drinking purposes in Mbarara city. There is a need for public health actors to improve the surveillance of microbiological quality of drinking water to minimize health risks.

Comprehensive identification of pathogenic microbes and antimicrobial resistance genes in food products using nanopore sequencing-based metagenomics.

Foodborne pathogens, particularly antimicrobial-resistant (AMR) bacteria, remain a significant threat to global health. Given the limitations of conventional culture-based approaches, which are limited in scope and time-consuming, metagenomic sequencing of food products emerges as a promising solution. This method provides a fast and comprehensive way to detect the presence of pathogenic microbes and antimicrobial resistance genes (ARGs). Notably, nanopore long-read sequencing provides more accurate bacterial taxonomic classification in comparison to short-read sequencing. Here, we revealed the impact of food types and attributes (origin, retail place, and food processing methods) on microbial communities and the AMR profile using nanopore metagenomic sequencing. We analyzed a total of 260 food products, including raw meat, sashimi, and ready-to-eat (RTE) vegetables. Clostridium botulinum, Acinetobacter baumannii, and Vibrio parahaemolyticus were identified as the top three foodborne pathogens in raw meat and sashimi. Importantly, even with low pathogen abundance, higher percentages of samples containing carbapenem and cephalosporin resistance genes were identified in chicken and RTE vegetables, respectively. In parallel, our results demonstrated that fresh, peeled, and minced foods exhibited higher levels of pathogenic bacteria. In conclusion, this comprehensive study offers invaluable data that can contribute to food safety assessments and serve as a basis for quality indicators.

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.

On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer.

The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets, and the horizontal transfer potential of extended-spectrum ß-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying ß-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a ß-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified: blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaPER-1. blaCTX-M-15 was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including sul1, str(A), str(B), erm(B), erm(F), intI1, aadA, incW, blaPSE, and blaOXA-20, were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however, erm(B) and erm(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for in vitro conjugation of plasmids conferring resistance to extended-spectrum ß-lactams and other classes of antibiotics into E. coli CV601. IMPORTANCE Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.

Comparison of the elimination effectiveness of tetracycline and AmpC ß-lactamase resistance genes in a municipal wastewater treatment plant using four parallel processes.

Municipal wastewater treatment plants (mWWTPs), considered reservoirs of antibiotic resistance genes (ARGs), are selected to compare the contributions of technology and process to ARG removal. Fifteen ARGs (tetA, tetB, tetC, tetE, tetG, tetL, tetM, tetO, tetQ, tetS, tetX, MOX, CIT, EBC, and FOX) and two integron genes (intI1, intI2) were tracked and detected in wastewater samples from a large-scale mWWTP with four parallel processes, including three biological technologies of AAO (anaerobic-anoxic-oxic), AB (adsorption-biodegradation), and UNITANK, two different disinfection technologies, and two primary sedimentation steps. The results showed that ARGs were widely detected, among which tetA and tetM had the highest detection rate at 100%. AAO was the most effective process in removing ARGs, followed by the AB and UNITANK processes, where the separation step was critical: 37.5% AmpC ß-lactamase genes were reduced by the secondary clarifier. UV disinfection was more efficient than chlorination disinfection by 47.0% in ARG removal. Both disinfection and primary sedimentation processes could effectively remove integrons, and the swirling flow grit chamber was a more effective primary settling facility in total ARG removal than the aerated grit chamber. The tet genes and AmpC ß-lactamase genes were significantly correlated with the water quality indexes of BOD5, CODCr, SS, TP, TOC, pH and NH4+-N (p < 0.05). In addition, the correlation between efflux pump genes and AmpC ß-lactamase genes was strongly significant (r2 = 0.717, p < 0.01). This study provides a more powerful guide for selecting and designing treatment processes in mWWTPs with additional consideration of ARG removal.

Prevalence of ß-Lactam Drug-Resistance Genes in Escherichia coli Contaminating Ready-to-Eat Lettuce.

Thirty-four Escherichia coli isolates from 91 ready-to-eat lettuce packages, obtained from local supermarkets in Northern California, were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for ß-lactamase genes. We found 15 distinct sequence types (STs). Six of these genotypes (ST1198, ST2625, ST2432, ST2819, ST4600, and ST5143) have been reported as pathogens found in human samples. Twenty-six (76%) E. coli isolates were resistant to ampicillin, 17 (50%) to ampicillin/sulbactam, 8 (23%) to cefoxitin, and 7 (20%) to cefuroxime. blaCTX-M was the most prevalent ß-lactamase gene, identified in eight (23%) isolates. We identified a class A broad-spectrum ß-lactamase SED-1 gene, blaSED, reported by others in Citrobacter sedlakii isolated from bile of a patient. This study found that fresh lettuce carries ß-lactam drug-resistant E. coli, which might serve as a reservoir for drug-resistance genes that could potentially be transmitted to pathogens that cause human infections.

An Integrative Database of ß-Lactamase Enzymes: Sequences, Structures, Functions, and Phylogenetic Trees.

ß-Lactamase enzymes have attracted substential medical attention from researchers and clinicians because of their clinical, ecological, and evolutionary interest. Here, we present a comprehensive online database of ß-lactamase enzymes. The current database is manually curated and incorporates the primary amino acid sequences, closest structural information in an external structure database (the Protein Data Bank [PDB]) and the functional profiles and phylogenetic trees of the four molecular classes (A, B, C, and D) of ß-lactamases. The functional profiles are presented according to the MICs and kinetic parameters that make them more useful for the investigators. Here, a total of 1,147 ß-lactam resistance genes are analyzed and described in the database. The database is implemented in MySQL and the related website is developed with Zend Framework 2 on an Apache server, supporting all major web browsers. Users can easily retrieve and visualize biologically important information using a set of efficient queries from a graphical interface. This database is freely accessible at http://ifr48.timone.univ-mrs.fr/beta-lactamase/public/.

Escherichia coli from Commercial Broiler and Backyard Chickens Share Sequence Types, Antimicrobial Resistance Profiles, and Resistance Genes with Human Extraintestinal Pathogenic Escherichia coli.

Escherichia coli recovered from poultry, and extraintestinal pathogenic E. coli (ExPEC), responsible for most cases of urinary tract infection (UTI) and bloodstream infection (BSI) in humans, may share genetic characteristics, suggesting that poultry are a potential source of ExPEC. Here, we compared E. coli isolated from commercial broiler and backyard chickens (n = 111) with ExPEC isolated from patients with community- or hospital-acquired UTI or BSI (n = 149) from Southeast Brazil. Isolates were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for ß-lactamase genes. We found that 10 genotypes were shared among poultry and human isolates: sequence type (ST) 10, ST48, ST58, ST88, ST90, ST93, ST131, ST602, ST617, and ST1018. Thirty-five (23%) ExPEC and 35 (31%) poultry E. coli isolates belonged to the shared STs. ST58 and ST88 isolates from human and poultry sources shared identical antimicrobial resistance profiles. blaTEM-1 was the most prevalent ß-lactamase gene, identified in 65 (92%) of 71 ExPEC and 29 (67%) of 43 poultry E. coli that tested positive for ß-lactamase genes. Commercial broiler chicken isolates shared the extended-spectrum ß-lactamase (ESBL) genes blaCTX-M-2,blaCTX-M-8, and blaSHV-2 with human isolates; backyard chicken isolates lacked ESBL genes. In conclusion, several genotypic and phenotypic characteristics were shared between human and poultry E. coli; this suggests that there is potential for transmission of E. coli and antimicrobial resistance genes from poultry to humans, perhaps through environmental contamination, direct contact, or consumption. Additional research is needed to understand the potential direction and pathways of transmission.

Epidemiology of healthcare-associated infections and mechanisms of antimicrobial resistance of responsible pathogens in Ukraine: a multicentre study.

BACKGROUND: Healthcare-associated infections (HAIs) caused by multidrug-resistant organisms (MDROs) have a high impact in terms of morbidity, mortality, and costs. AIM: To estimate the prevalence and incidence of HAIs, and to describe phenotypic and genotypic features of antimicrobial resistance in responsible pathogens in Ukraine. METHODS: Prospective multicentre surveillance was conducted from January 2019 to December 2021 in 17 regional hospitals of Ukraine. Definitions of HAIs were adapted from the Centers for Disease Control and Prevention's National Healthcare Safety Network. FINDINGS: Among 37,968 patients, 6218 (16.4%) HAIs were observed. Of all HAI cases, 14.8% were detected after hospital discharge. The most frequently reported HAI types were pneumonia (24.4%), urinary tract infections (19.8%), surgical site infections (15.3%), and bloodstream infections (11.2%). Of all HAIs, 11.9% were defined as part of an outbreak. Death during hospitalization was reported in 12.6% of HAI cases. In total, 85.1% isolates from patients were found to be MDROs. Meticillin resistance was found in 41.2% of S. aureus (MRSA) isolates, and vancomycin resistance was found in 11.8% of enterococci. Antimicrobial resistance to third-generation cephalosporins was detected in 48.4% of all Enterobacterales. Antimicrobial resistance to carbapenems was detected in 71.3% of all non-fermentative Gram-negative bacteria. Of the all isolates tested, 25.1% were found to be multidrug-resistant (MDR). CONCLUSION: This study found a high prevalence of HAIs; those caused by MDROs varied widely depending on the bacterial species, antimicrobial group, and geographical region of Ukraine. MDROs were one of the main causes of HAI-associated deaths.

Antimicrobial Resistance Linked to Septic System Contamination in the Indiana Lake Michigan Watershed.

Extended-spectrum ß-lactamases confer resistance to a variety of ß-lactam antimicrobials, and the genes for these enzymes are often found on plasmids that include additional antimicrobial resistance genes (ARG). We surveyed aquatic environments in the Indiana Lake Michigan watershed in proximity to areas with high densities of residential septic systems to determine if human fecal contamination from septic effluent correlated with the presence of antimicrobial resistance genes and phenotypically resistant bacteria. Of the 269 E. coli isolated from environmental samples and one septic source, 97 isolates were resistant to cefotaxime, a third-generation cephalosporin. A subset of those isolates showed phenotypic resistance to other ß-lactams, fluoroquinolones, sulfonamides, and tetracyclines. Quantitative PCR was used to quantify human-associated Bacteroides dorei gene copies (Human Bacteroides) from water samples and to identify the presence of ARG harbored on plasmids from E. coli isolates or in environmental DNA. We found a strong correlation between the presence of ARG and human fecal concentrations, which supports our hypothesis that septic effluent is a source of ARG and resistant organisms. The observed plasmid-based resistance adds an additional level of risk, as human-associated bacteria from septic systems may expand the environmental resistome by acting as a reservoir of transmissible resistance genes.

Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission?

IMPORTANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) sequence type (ST) 38 is one of the top 10 human pandemic lineages. Although a major cause of urinary tract and blood stream infections, ST38 has been poorly characterized from a global phylogenomic perspective. A comprehensive genome-scale analysis of 925 ST38 isolate genomes identified two broad ancestral clades and linkage of discrete ST38 clusters with specific bla CTX-M variants. In addition, the clades and clusters carry important virulence genes, with diverse but poorly characterized plasmids. Numerous putative interhost and environment transmission events were identified here by the presence of ST38 clones (defined as isolates with ≤35 SNPs) within humans, companion animals, food sources, urban birds, wildlife, and the environment. A small cluster of international ST38 clones from diverse sources, likely representing progenitors of a hospital outbreak that occurred in Brisbane, Australia, in 2017, was also identified. Our study emphasizes the importance of characterizing isolate genomes derived from nonhuman sources and geographical locations, without any selection bias.

Intestinal loads of extended-spectrum beta-lactamase and Carbapenemase genes in critically ill pediatric patients.

Introduction: Intestinal colonization by Multi-Drug Resistant Organisms (MDROs) can pose a threat on the health of critically ill patients. The extent of colonization by these organisms is related to previous antibiotic treatments and their ability to cause infections among adult patients. The aim of this study is to determine the relationship between the intestinal Relative Loads (RLs) of selected antibiotic resistance genes, antibiotic consumption and extra-intestinal spread among critically ill pediatric patients. Methods: RLs of bla CTX-M-1-Family, bla OXA-1, bla OXA-48 and bla VIM were determined in 382 rectal swabs obtained from 90 pediatric critically ill patients using qPCRs. The RLs were compared to the patients' demographics, antibiotic consumption, and detection of MDROs from extra-intestinal sites. 16SrDNA metagenomic sequencing was performed for 40 samples and clonality analyses were done for representative isolates. Results and discussion: 76 (74.45%) patients from which 340 (89.01%) rectal swabs were collected had at least one swab that was positive for one of the tested genes. Routine cultures did not identify carbapenemases in 32 (45.1%) and 78 (58.2%) swabs that were positive by PCR for bla OXA-48 and blaVIM, respectively. RLs of above 6.5% were associated with extra-intestinal spread of blaOXA-48-harboring MDROs. Consumption of carbapenems, non-carbapenem ß-lactams, and glycopeptides were statistically associated with testing negative for bla CTX-M-1-Family and bla OXA-1 while the consumption of trimethoprim/sulfamethoxazole and aminoglycosides was associated with testing negative for blaOXA-48 (P<0.05). In conclusion, targeted qPCRs can be used to determine the extent of intestinal dominance by antibiotic resistant opportunistic pathogens and their potential to cause extra-intestinal infections among a critically ill pediatric population.

Detection of AmpC ß-lactamases in gram-negative bacteria.

AmpC ß-lactamase genes are clinically important because they often confer resistance to most ß-lactams other than 4th-generation cephalosporins and carbapenems. However, traditional and existing detection methods are expensive, labor-intensive and range-limited. We established an efficient multiplex PCR method to simultaneously identify six families of ampC ß-lactamase genes, ACC, EBC, CIT, DHA, MOX and FOX, and evaluated the sensitivity and specificity of this assay. The multiplex method could accurately identify ACC, EBC, CIT, DHA, MOX and FOX variants among a total of 175 ampC ß-lactamase genes. The minimum concentration of genomic DNA that could be detected was 1.0×103 copies/µL. We subsequently used this method to analyze 2 Salmonella spp. with carrying CMY-2 and DHA-1, and 167 Enterobacteriaceae isolates in blinded PCR testing. Positive isolates produced bright bands that corresponded with their genotype. Results were in concordance with those of the traditional method but showed increased sensitivity and accuracy. This indicates that the newly developed multiplex PCR system could be used as a diagnostic tool to accurately distinguish the six families of ampC ß-lactamase genes with high efficiency, wide range, easy operation and good discrimination.

Characterization of Acinetobacter baumannii Isolated from Raw Milk.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen associated with nosocomial infections. In this study, 100 raw milk samples were collected from Qena, Egypt, and subjected to conventional and molecular assays to determine the presence of A. baumannii and investigate their antimicrobial resistance and biofilm formation. Our findings revealed that, among the 100 samples, Acinetobacter spp. were found in 13 samples based on CHROM agar results. We further characterized them using rpoB and 16S-23SrRNA sequencing and gyrB multiplex PCR analysis and confirmed that 9 out of the 13 Acinetobacter spp. isolates were A. baumannii and 4 were other species. The A. baumannii isolates were resistant to ß-lactam drugs, including cefotaxime (44%), ampicillin-sulbactam and levofloxacin (33.3% for each), imipenem, meropenem and aztreonam (22.2% for each). We observed different antimicrobial resistance patterns, with a multi-antibiotic resistant (MAR) index ranging from 0.2 to 0.3. According to the PCR results, blaOXA-51 and blaOXA-23 genes were amplified in 100% and 55.5% of the A. baumannii isolates, respectively, while the blaOXA-58 gene was not amplified. Furthermore, the metallo-ß-lactamases (MBL) genes blaIMP and blaNDM were found in 11.1% and 22.2% of isolates, respectively, while blaVIM was not amplified. Additionally, eight A. baumannii isolates (88.8%) produced black-colored colonies on Congo red agar, demonstrating their biofilm production capacity. These results showed that, besides other foodborne pathogens, raw milk should also be examined for A. baumannii, which could be a public health concern.

Antimicrobial resistance genes in microbiota associated with sediments and water from the Akaki river in Ethiopia.

The spread of antimicrobial-resistant pathogens is a global health concern. Most studies report high levels of antimicrobial resistance genes (ARGs) in the aquatic environment; however, levels associated with sediments are limited. This study aimed to investigate the distribution of ARGs in the sediments and water of the Akaki river in Addis Ababa, Ethiopia. The diversity and abundance of 84 ARGs and 116 clinically important bacteria were evaluated from the sediments and water collected from five sites in the Akaki river. Most of the ARGs were found in the city close to anthropogenic activities. Water samples collected in the middle catchment of the river contained 71-75% of targeted ARGs, with genes encoding aminoglycoside acetyltransferase (aac(6)-Ib-cr), aminoglycoside adenylyl transferase (aadA1), ß-lactamase (blaOXA-10), quinolone resistance S (qnrS), macrolide efflux protein A (mefA), and tetracycline resistance (tetA), were detected at all sampling sites. Much fewer ARGs were detected in all sediments, and those near the hospitals had the highest diversity and level. Despite the lower levels and diversity, there were no unique ARGs detected in the sediments that were also not detected in the waters. A wide range of clinically relevant pathogens were also detected in the Akaki river. The findings suggest that the water phase, rather than the sediments in the Akaki river, is a potential conduit for the spread of ARGs and antibiotic-resistant bacteria.

Intestinal Dominance by Multidrug-Resistant Bacteria in Pediatric Liver Transplant Patients.

Pediatric liver transplantation (PLTx) is commonly associated with extensive antibiotic treatments that can produce gut microbiome alterations and open the way to dominance by multidrug-resistant organisms (MDROs). In this study, the relationship between intestinal Relative Loads (RLs) of ß-lactamase genes, antibiotic consumption, microbiome disruption, and the extraintestinal dissemination of MDROs among PLTx patients is investigated. 28 PLTx patients were included, from whom 169 rectal swabs were collected. Total DNA was extracted and blaCTX-M-1-Family, blaOXA-1, blaOXA-48, and blaVIM were quantified via quantitative polymerase chain reaction (qPCR) and normalized to the total bacterial load (16SrRNA) through LogΔΔCt to determine the RLs. 16SrRNA sequencing was performed for 18 samples, and metagenomic sequencing was performed for 2. Patients' clinical data were retrieved from the hospital's database. At least one of the genes tested were detected in all of the patients. The RLs for blaCTX-M-1-Family, blaOXA-1, blaOXA-48, and blaVIM were higher than 1% of the total bacterial population in 67 (80.73%), 56 (78.87%), 57 (77.03%) and 39 (61.9%) samples, respectively. High RLs for blaCTX-M-1-Family, blaOXA-1, and/or blaOXA-48, were positively associated with the consumption of carbapenems with trimethoprim-sulfamethoxazole and coincided with low diversity in the gut microbiome. Low RLs were associated with the consumption of noncarbapenem ß-lactams with aminoglycosides (P < 0.05). Extraintestinal isolates harboring the same gene(s) as those detected intraintestinally were found in 18 samples, and the RLs of the respective swabs were high. We demonstrated a relationship between the consumption of carbapenems with trimethoprim-sulfamethoxazole, intestinal dominance by MDROs and extraintestinal spread of these organisms among PLTx patients. IMPORTANCE In this study, we track the relative intestinal loads of antibiotic resistance genes among pediatric liver transplant patients and determine the relationship between this load, antibiotic consumption, and infections caused by antibiotic-resistant organisms. We demonstrate that the consumption of broad spectrum antibiotics increase this load and decrease the gut microbial diversity among these patients. Moreover, the high loads of resistance genes were related to the extraintestinal spread of multidrug-resistant organisms. Together, our data show that the tracking of the relative intestinal loads of antibiotic resistance genes can be used as a biomarker that has the potential to stop the extraintestinal spread of antibiotic-resistant bacteria via the measurement of the intestinal dominance of these organisms, thereby allowing for the application of preventive measures.

Multiple antibiotic resistance and DNA methylation in Enterobacteriaceae isolates from different environments.

Antibiotic resistant bacteria with diverse resistance phenotypes and genotypes are ubiquitous in the environments that have become a global health concern. The role of DNA methylation in the dissemination of antibiotic resistance among different environments is currently unclear. We recovered 646 Enterobacteriaceae (Eb) isolates from hospital, livestock manure, municipal wastewater-treatment plants, river sediment and soil for comprehensive analysis of resistance phenotypes, ß-lactamase genes, integrons, integron-associated gene cassettes and the levels of DNA methylation. Antibiotic susceptibility testing revealed that approximately 87.31 % isolates were multidrug resistant Eb. The ß-lactamase genes were positively detected in 473 isolates with greater diversity in human or animal sourced Eb, while its prevalence was found to be highest in the Eb isolates from the natural environments. Forty-three gene cassettes (28 different types mediated by intI1) were detected in 53 (19.63 %) isolates, with greater diversity in Eb isolates from hospital and livestock manure. The multiple antibiotic resistance index of single strain was positively correlated with the 5-methylcytosine and showed a negative correlation with 6-methylademine. We conclude that the development of antibiotic resistance could possibly be coupled with DNA methylation, which might enhance the antimicrobial resistance and survival capacity of Eb.

Prevalence, antimicrobial resistance, and genotyping of Shiga toxin-producing Escherichia coli in foods of cattle origin, diarrheic cattle, and diarrheic humans in Egypt.

Shiga toxin-producing Escherichia coli (STEC) is a pathotype of E. coli that causes enteric and systemic diseases ranging from diarrhoea to severe hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The emergence of multidrug-resistant (MDR) STEC from cattle sources has increased public health risk and limited treatment options. The prevalence of STEC was investigated in 200 raw food samples (milk and beef samples) and 200 diarrheic samples (cattle and human samples) in a matched region. The presence of stx genes (stx1 and stx2), carbapenemase-encoding genes (blaVIM, blaNDM-1, and blaIMP), and extended-spectrum ß-lactamase (ESBL)-encoding genes (blaTEM group, blaCTX-M1 group, and blaOXA-1 group) was screened by polymerase chain reaction (PCR). Antibiogram and Enterobacterial repetitive intergenic consensus (ERIC)-PCR were also conducted. STEC isolates were identified in 6.5% (13/200) of food samples [6% (6/100) of milk and 7% (7/100) of beef samples] and in 11% (22/200) of diarrheic cases [12% (12/100) of cattle and 10% (10/100) of human samples]. We found that O26 (4.5%, 18/400) and O111 (1.5%, 6/400) were the most prevalent STEC serovars and were found more commonly in diarrheic samples. STEC strains with both stx genes, stx2 only, and stx1 only genotypes were present in 62.9% (22/35), 20% (7/35), and 17.1% (6/35) of isolates, respectively. Carbapenemase-producing STEC (CP STEC) isolates were found in 1.8% (7/400) of samples [0.5% (1/200) of foods and 3% (6/200) of diarrheic cases]. The blaVIM gene was detected in all CP STEC isolates, and one human isolate carried the blaNDM-1 gene. ESBL-producing STEC strains were detected in 4.3% (17/400) of samples [1.5% (3/200) of food samples and 7% (14/200) of diarrheic cases]. The blaTEM, blaCTX-M1, and blaOXA-1 genes were detected in 42.9% (15/35), 28.6% (10/35), and 2.9% (1/35) of STEC isolates, respectively. Approximately half (51.4%, 18/35) of STEC isolates were MDR STEC; all CP STEC and ESBL-producing STEC were also MDR STEC. The highest antimicrobial resistance rates were found against nalidixic acid (51.4%) and ampicillin (48.6%), whereas the lowest rates were reported against gentamicin (5.7%) and ciprofloxacin (11.4%). MDR STEC strains were 5.3 times more likely to be found in diarrheic cases than in foods (P = 0.009, 95% CI 1.5-18.7). ERIC-PCR was used for genotyping STEC isolates into 27 different ERIC-types (ETs) with a discrimination index of 0.979. Five ETs showed clusters of 2-4 identical isolates that shared the same virulence and antibiotic resistance genetic profile. Human isolates matched food isolates in two of these ET clusters (the O26 CP STEC cluster and the O111 STEC cluster), highlighting the potential cross-species zoonotic transmission of these pathogens and/or their genes in the study region. This is the first detection of CP STEC in milk and diarrheic cattle in Egypt.

Molecular Diversity of ESBL-Producing Escherichia coli from Foods of Animal Origin and Human Patients.

Dissemination of enterobacteria that produce extended spectrum ß-lactamases (ESBL) throughout the food chain has become an important health concern. This work aimed to evaluate the occurrence of ESBL-producing bacteria in foods of animal origin and to investigate the similarities between food and human isolates. The presence of beta-lactam-resistant Enterobacteriaceae was analyzed in 108 food samples, isolating 10 strains of Escherichia coli, one strain of Citrobacter freundi, and one of Hafnia alvei. E. coli isolates were compared to a group of 15 strains isolated from human patients by antibiotic susceptibility testing, characterization of ESBL genes (blaTEM, blaCTX,), multilocus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE). Nineteen (14 clinical and five food) isolates carried blaCTX, 14 (six clinical and eight food) carried blaTEM, and three (one clinical and two food) carried blaSHV gen. MLST analysis revealed the prevalence of ST131 among the clinical strains, which grouped together in a PFGE cluster. Food isolates showed higher diversity and two of them (ST57) grouped with clinical strains, whereas another two belonged to clonal groups with virulence potential (ST59). In conclusion, the results showed that foods of animal origin must be regarded as a reservoir of ESBL-producing bacteria of clinical relevance, which might spread through the food chain.

Fourth Generation Cephalosporin Resistance Among Salmonella enterica Serovar Enteritidis Isolates in Shanghai, China Conferred by bla CTX-M-55 Harboring Plasmids.

In this study, we investigated the pattern of antimicrobial resistance in Salmonella enterica serotype Enteritidis isolates in Shanghai, China from 2005 to 2014. We found the first isolates with resistance to the fourth-generation cephalosporin cefepime starting in 2010. Furthermore, we analyzed the epidemic characteristics and mechanisms of underlying cefepime resistance in S. Enteritidis isolates found from 2010. In total, 38 of 2,914 (1.30%) isolates were identified as cefepime-resistant S. Enteritidis (CRSE) isolates by Kirby-Bauer disk diffusion. Two isolates were from animal derived food sources; 36 isolates were from fecal samples of human patients with salmonellosis. Antimicrobial susceptibility testing using the agar dilution method revealed that all CRSE isolates showed additional resistances at least to ceftazidime, cefotaxime, and ampicillin. Additionally, pulsed-field gel electrophoresis (PFGE) profiles indicated that 89.47% of CRSE isolates also displayed similar PFGE patterns. Five types of ß-lactamase genes, bla CTX-M (100.00%, 38/38), bla SHV (65.79%, 25/38), bla TEM (52.63%, 20/38), bla ACC (18.42%, 7/38), and bla PSE (5.26%, 2/38) were detected by PCR and sequencing. Among bla CTX-M genes, bla CTX-M-55 was the dominant type (84.21%, 32/38). Conjugation and transformation experiments along with plasmid replicon typing revealed that bla CTX-M-55 was located on plasmids of various replicon types with sizes ranging from 76.8 to 138.9 kb. Plasmid sequence analysis also showed that the bla CTX-M-55 gene was mobilized mainly by the ISEcp1-bla CTX-M-55-ORF477 transposition unit and had its own ISEcp1-based promoter, which accelerated the expression and transmission of bla CTX-M-55. Analysis of whole genome sequences (Illumina) of one selected transformant SH12G706-C showed high similarity of the bla CTX-M-55 carrying plasmid with the IncI1 plasmid backbone p628-CTX-M of Klebsiella pneumoniae detected in 2010 in China. The present study demonstrated that the bla CTX-M-55 gene mobilized by ISEcp1- bla CTX-M-55-ORF477 was the main feature shared by CRSE isolates and seems to play an important role for transmission of cefepime resistance. The number of CRSE isolates is rising annually, and the strong dissemination ability of ISEcp1-bla CTX-M-55-ORF477-harboring plasmids among different species represents an important threat to the therapeutic effectiveness of cefepime.