Β-lactamase Genes Research Articles

Antimicrobial Susceptibility and Characterization of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Stools of Primary Healthcare Patients in Ethiopia.

Antimicrobial resistance of Escherichia coli is a growing problem in both developed and developing countries. This study aimed to investigate the phenotypic antimicrobial resistance of E. coli isolates (n = 260) isolated from the stool specimen of patients attending public health facilities in Addis Ababa and Hossana. This study also aimed to characterize phenotypically confirmed extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates (n = 22) using whole-genome sequencing. Resistance to 18 different antimicrobials was assessed using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The highest resistance rate among the E. coli isolates was found for ampicillin (52.7%), followed by trimethoprim-sulfamethoxazole (29.6%). Of all isolates, 50 (19.2%) were multidrug-resistant and 22 (8.5%) were ESBL producers. ESBL genes were detected in 94.7% of the sequenced E. coli isolates, and multiple β-lactamase genes were detected in 57.9% of the isolates. The predominant ESBL gene identified was blaCTX-M-15 (78.9%). The blaTEM-1B gene was detected in combination with other ESBL genes in 57.9% of the isolates, while only one of the sequenced isolates contained the blaTEM-1B gene alone. The blaCTX-M-3 gene was detected in three isolates. The genes blaCTX-M-15 and blaTEM-1B as well as blaCTX-M-15 and blaTEM-169 were confirmed to coexist in 52.6% and 10.5% of the sequenced E. coli isolates, respectively. In addition, blaOXA-1 was identified together with blaCTX-M-15 and blaTEM-1B in one isolate, and in one isolate, blaTEM-169 together with blaCTX-M-15 and blaTEM-1B was found. The results obtained show that measures need to be taken to reduce the spread of drug resistance and ensure the long-term use of available antimicrobials.

Occurrence and characteristics of extended-spectrum-β-lactamase- and pAmpC-producing Klebsiella pneumoniae isolated from companion animals with urinary tract infections.

This study examined 70 Klebsiella pneumoniae isolates derived from companion animals with urinary tract infections in Taiwan. Overall, 81% (57/70) of the isolates carried extended-spectrum β-lactamase (ESBL) and/or plasmid-encoded AmpC (pAmpC) genes. ESBL genes were detected in 19 samples, with blaCTX-M-1, blaCTX-M-9, and blaSHV being the predominant groups. pAmpC genes were detected in 56 isolates, with blaCIT and blaDHA being the predominant groups. Multilocus sequence typing revealed that sequence types (ST)11, ST15, and ST655 were prevalent. wabG, uge, entB, mrkD, and fimH were identified as primary virulence genes. Two isolates demonstrated a hypermucoviscosity phenotype in the string test. Antimicrobial susceptibility testing exhibited high resistance to β-lactams and fluoroquinolones in ESBL-positive isolates but low resistance to aminoglycosides, sulfonamides, and carbapenems. Isolates carrying pAmpC genes exhibited resistance to penicillin-class β-lactams. These findings provide valuable insights into the role of K. pneumoniae in the context of the concept of One Health.

PREVALENCE OF BLATEM, BLASHV, AND BLACTX-M GENES AMONG ESBL-PRODUCING ESCHERICHIA COLI ISOLATED FROM THE BLOOD SAMPLES OF ICUS PATIENTS OF UNIVERSITY HOSPITALS IN SANA'A CITY, YEMEN

Aim and Objective: With the emergence of organisms such as Enterobacteriaceae that produce extended-spectrum β-lactamase (ESBL), which are resistant to multiple medications (multidrug-resistance), concerns about how best to treat infections have significantly increased. The current study examined the molecular features of ESBL in clinical isolates of Escherichia coli that resulted in bloodstream infections as well as the pattern of antibiotic resistance to gather useful data on the infection's epidemiology among Yemeni ICU patients. Subjects and methods: A cross-sectional study was conducted on sepsis patients admitted in intensive care units at four hospitals in Sana'a, Yemen, between January, 2021 and April, 2022. Blood cultures were used on patients suspected of having sepsis. Standard laboratory procedures were then used to isolate and identify possible bacterial infections, and the disk diffusion method was used to test for microbial susceptibility. All strains were tested for Extended Spectrum Beta-Lactamase (ESBL) production using the Modified Double Disc Synergy Test (MDDST). Following analysis, polymerase chain reaction, β-lactamase genes (blaTEM, blaSHV, and blaCTX-M) were identified. Results: The results of the conventional PCR experiment revealed that 33.3% of the blaCTX-M genes, 0.0% of blaSHV, and 100% of blaTEM were present in the strains of ESBL-producing E. coli that were collected. It was discovered that the E. coli isolates' patterns of antibiotic resistance to 23 different antibiotics differed greatly. The bulk of the E. coli isolates were found to be multi-drug resistant (MDR). Furthermore, MDR characteristics were observed in 85% of E. coli isolates. Conclusion: Control and surveillance of antibiotic resistance depend on an understanding of the resistance genes and patterns of antimicrobial resistance of bacterial pathogens within a given geographic area. The current study's findings showed that MDR was very common. According to the current study's findings, TEM was significantly more common than other ESBL gene types. Peer Review History: Received: 7 November 2023; Revised: 12 December; Accepted: 6 January 2024, Available online: 15 January 2024 Academic Editor: Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, amaka_mgbahurike@yahoo.com Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.5/10 Average Peer review marks at publication stage: 8.0/10 Reviewers: Dr. Rola Jadallah, Arab American University, Palestine, rola@aauj.edu Dr. Wadhah Hassan Ali Edrees, Hajja University, Yemen, edress2020@gmail.com

Antimicrobial resistance profiles of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae strains isolated from broiler chickens

Globally, the spread of multidrug-resistant Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from food to humans poses a severe threat to public health. The aim of this study was to assess the co-occurrence of colistin and β-lactamase resistance genes in E. coli, K. pneumoniae, and P. aeruginosa strains isolated from faeces of abattoir broiler chickens. The E. coli, P. aeruginosa and K. pneumoniae isolates were successfully detected from faecal samples by polymerase chain reaction (PCR) at infection rates of 60.7%, 22.5% and 16.7% respectively. The isolates displayed the highest levels of antibiotic resistance (AR) against ampicillin (82.3%) and amoxicillin-clavulanic acid (74.2%) for E. coli, followed by cefoxitin (70.6%) for K. pneumoniae, whilst P. aeruginosa displayed 26.1% antibiotic resistance (AR) against both ampicillin and colistin sulphate. The colistin mcr-1 gene was harboured by 46.8%, 47.1% and 21.7%, E. coli, K. pneumonia and P. aeruginosa isolates respectively. Ten out of 62 (16.1%), 6/17 (35.3%), 4/23 (17.4%) isolates were phenotypically classified as ESBL E. coli, K. pneumoniae, and P. aeruginosa respectively. The ESBL-E. coli isolates respectively possessed blaCTX-M (60%), blaTEM (20%) and blaCTX-M-9 (10%) genes. The ESBL-K. pneumoniae harboured, blaCTX-M (50%), blaOXA (33%), blaCARB (17%), and blaCTX-M-9 (17%) genes respectively, whilst, P. aeruginosa isolates respectively carried blaTEM (75%), blaCTX-M (50%), blaOXA (25%) and blaCARB (25%) genes. Molecular analysis identified the blaCTX-Mβ-lactamase-encoding genes collectively from E. coli, P. aeruginosa, K. pneumoniae isolates. Colistin and β-lactamase genes were present in only 16.7%, 6.9%, and 2.9% of E. coli, K. pneumoniae, and P. aeruginosa isolates, respectively. A total of 17, 7 and 3 isolates for E. coli, K. pneumoniae and P. aeruginosa respectively carried both colistin and β-lactamase antibiotics resistant genes. This is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Data generated from this study will contribute to formulation of new strategies for combating spread of E. coli, K. pneumoniae, and P. aeruginosa isolates as well as prevention of their AR development.

A preliminary exploration on the mechanism of the carbapenem-resistance transformation of Serratia marcescens in vivo

BackgroundThe infection of carbapenem-resistant organisms was a huge threat to human health due to their global spread. Dealing with a carbapenem-resistant Serratia marcescens (CRSM) infection poses a significant challenge in clinical settings. This study aims to provide insights into strategies for controlling CRSM infection by exploring the transformation mechanism of carbapenem-resistance.MethodsWe used whole genome sequencing (WGS) to investigate the mechanism of carbapenem resistance in 14 S. marcescens isolates in vivo. The expression level of related genes and the minimum inhibitory concentration of meropenem (MICMEM) were also evaluated to confirm the mechanism of carbapenem resistance.ResultsSeven groups of S. marcescens, each consisting of two strains, were collected from a hospital and displayed a shift in MICMEM from low to high levels. Homology analysis revealed that the isolates in five groups were significantly different from the remaining two. WGS and experimental evidence indicated that four groups of strains developed carbapenem resistance by acquiring the blaKPC (obtaining group), while two groups (persisting group) increased the expression level of the blaKPC. In contrast, isolates in the last group (missing group) did not carry the blaKPC. All strains possessed multiple β-lactamase genes, including blaCTX−M−14, blaSRT−1, and blaSRT−2. However, only in the missing group, the carbapenem-resistant strain lost an outer membrane protein-encoding gene, leading to increased blaCTX−M−14 expression compared to the carbapenem-susceptible strain.ConclusionThe study findings suggest that S. marcescens strains developed diverse carbapenem resistance in vivo through the evolution of drug resistance, rather than through clone replacement. We hypothesize that carbapenem resistance in S. marcescens was due to certain clonal types with a distinct mechanism.

Molecular characterization of antimicrobial resistance genes of Staphylococcus aureus isolated from mastitic camel milk in Egypt.

Staphylococcus aureus is one of the most common causes of mastitis worldwide. This study aimed to determine the prevalence and antimicrobial resistance (AMR) patterns of S. aureus in mastitic milk samples collected from camel farms in Matrouh Governorate, Egypt. A total of 200 mastitic camel milk samples were evaluated for S. aureus using both conventional culture-based and molecular-based methods. Antibiotic susceptibility testing of S. aureus isolates was conducted using disc diffusion and agar dilution methods, with antibiotic resistance genes identified through polymerase chain reaction with specific primers. Out of samples tested, 60 (30.00%) were positive for S. aureus. The isolates displayed the highest of resistance against piperacillin-tazobactam (55.00%) followed by trimethoprim- sulfamethoxazole (45.00%) and amoxicillin (40.00%). Half of the isolates were multidrug-resistant (MDR). The AMR genes included methicillin-resistant gene (mecA), β-lactamase gene (blaZ), tetracycline resistance gene (tetK), erythromycin resistance gene (ermB) and vancomycin resistant gene (vanA) were detected in 100%, 100%, 95.00%, 90.00% and 20.00% of the isolates, respectively. In conclusion, the presence of MDRS aureus as a cause of clinical camel mastitis is a significant veterinary and public health concern. These findings highlight the importance of proper antibiotic use in Egyptian camel farms and the need for molecular techniques to fully understand the genetic profile of antimicrobial-resistant S. aureus isolates.

Identification and characterization of a plasmid co-harboring blaCTX-M-55 and blaTEM-141 in Escherichia albertii from broiler in China1

The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials. In this study, we isolated four Escherichia albertii (E. albertii) strains from broilers in Eastern China. The antimicrobial susceptibility and genomic characterization of these E. albertii isolates were determined. Our results revealed that these four E. albertii isolates exhibited resistance to tetracyclines, chloramphenicol, β-lactams, aminoglycosides, polymyxin B, sulfonamides, quinolones, and other antimicrobials. Among them, EA04 isolate was multidrug resistant and harbored extended-spectrum β-lactamases (ESBL) genes blaCTX-M and blaTEM. Whole genome sequencing and core-genome Multilocus sequence typing (cgMLST) based on all ST4638 E. albertii for EA04 inferred highly probable epidemiological links between selected human isolates. Additionally, the ESBL genes blaTEM-141 and blaCTX-M-55 were coexistent in an approximately 75 kb IncFII plasmid pEA04.2 in EA04. Comparative analysis indicated that genes blaTEM-141 and blaCTX-M-55 were located in IS15-blaCTX-M-55-wbuC-blaTEM-141-IS26 region, which similar structures were identified in various bacteria. Furthermore, the plasmid pEA04.2 could be transferable to Escherichia coli EC600 and lead to the resistance to third-generation cephalosporins. These results suggested that chicken potentially serve as a reservoir for multidrug resistant E. albertii, which increases the risk of horizontal transfer of antimicrobial resistance between humans, animals and environment.

Genetic characterization of virulence and extended spectrum β-lactamase producing genes of Klebsiella pneumoniae isolated from bovine milk.

Mastitis associated Klebsiella pneumoniae species were isolated from bovine milk to characterize virulence genes (wabG and kfuBC) and extended spectrum β-lactamase (ESBL) genes (blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaTEM, blaSHV and blaOXA). A total number of 325 bovine milk samples (195 raw and 130 mastitic milk specimens) collected from Banaskantha, a milk-shed district of Gujarat, India, were included in the study. A total number of 27 K. pneumoniae isolates were recovered, consisting of 17 (62.96%) isolates from raw milk and 10 (37.03%) isolates from mastitic milk samples, giving an overall prevalence of 8.31%. Antibiotic sensitivity patterns revealed that 20 out of 27 isolates were found to be multi-drug resistant. Based on combination disc diffusion test and HiCrome ESBL agar method, 20 (74.07%) and 25 (92.59%) isolates were detected as ESBL producers, respectively. Among virulence genes studied, presence of wabG (25/27; 92.59%) was higher than kfuBC (5/27; 18.51%). Beta-lactamase genes viz., blaSHV, blaTEM and blaCTX-M-1 were detected in 23/27 (85.18%), 3/27 (11.11%) and 2/27 (7.40%) of isolates, respectively; while, none of the isolates was found to be positive for blaCTX-M-9 and blaOXA-1 genes. Outcome of the study provided an insight into virulence genes and ESBL producing K. pneumoniae isolated from bovine milk samples in India.

Multi-drug resistant (MDR) Gram-negative pathogenic bacteria isolated from poultry in the Noakhali region of Bangladesh.

Rapidly increasing antibiotic-resistant bacterial strains in Bangladesh's food and farm animals stem from the excessive and inappropriate use of antibiotics. To assess the prevalence of multi-drug resistant (MDR) Gram-negative bacteria in poultry chicks, we sought to isolate and identify strains carrying antimicrobial resistance genes. Isolation and identification involved biochemical tests, 16S rRNA sequencing, and PCR screening of species-specific genes. MDR patterns were evaluated using CLSI guidelines with seventeen antibiotics across twelve classes. Targeted gene sequences were amplified for the detection of Extended-spectrum β-Lactamase (ESBL), carbapenem, tetracycline, sulfonamide, and colistin resistance genes. Common isolates, such as Escherichia coli, Klebsiella pneumoniae, Proteus penneri, and Enterobacter hormaechei, exhibited average Multiple Antimicrobial Resistance (MAR) indices of 0.66, 0.76, 0.8, 0.84, and 0.81, 0.76, 0.84, 0.41 for broiler and layer chicken, respectively. Providencia stuartii and Salmonella enterica, exclusive to broiler samples, had MAR indices of 0.82 and 0.84, respectively. Additional isolates Morganella morganii, Aeromonas spp., and Wohlfahrtiimonas chitiniclastica were found in layers (Average MAR indices: 0.73, 0.71, and 0.91). Notably, M. morganii, E. hormaechei and W. chitiniclastica were identified for the first time in Bangladeshi poultry chicken, although their evolution is yet to be understood. In this study, Pan-drug resistance was observed in one P. stuartii (broiler) and one Aeromonas spp. (layer) with a MAR index 1, while all isolates exhibited MAR indices >0.2, indicating MDR. Antimicrobial resistance (AMR) gene screening identified blaTEM, blaSHV, tetA, and sul1 in a majority of the MDR strains. Interestingly, E. coli (lactose positive and negative) and E. hormaechei were exclusively found to possess the tetB gene. In addition, E. coli (lactose negative), Klebsiella pneumoniae, Enterobacter hormaechei, M. morganii, and P. stuartii were observed to carry the colistin-resistant mcr-1 gene, whereas sul2 was detected in E. coli (lactose positive and negative), E. hormaechei, P. stuartii, and P. penneri. These findings emphasize the health risk of our consumers of both broiler and layer chickens as they have turned into a potent reservoir of various AMR gene carrying MDR and Pan-drug resistant bacteria.

Molecular Detection of β-lactamase Genes and Antibiotics Resistance for Bacterial Isolates From Pigeon’s Eyes Infections

Molecular Detection of β-lactamase Genes and Antibiotics Resistance for Bacterial Isolates From Pigeon’s Eyes Infections

Genomic characterization of extended-spectrum β-lactamase-producing Enterobacterales isolated from abdominal surgical patients.

Rectal swabs of 104 patients who underwent abdominal surgery were screened for ESBL producers. Sequence types (STs) and resistance genes were identified by whole-genome sequencing of 46 isolates from 17 patients. All but seven isolates were assigned to recognized STs. While 18 ESBL-producing E. coli (EPEC) strains were of unique STs, ESBL-producing K. pneumoniae (EPKP) strains were mainly ST14 or ST15. Eight patients harboured strains of the same ST before and after abdominal surgery. The most prevalent resistant genes in E. coli were blaEC (69.57%), blaCTX-M (65.22%), and blaTEM (36.95%), while blaSHV was present in only K. pneumoniae (41.30%). Overall, genes encoding β-lactamases of classes A (blaCTX-M, blaTEM, blaZ), C (blaSHV, blaMIR, and blaDHA), and D (blaOXA) were identified, the most prevalent variants being blaCTX-M-15, blaTEM-1B, blaSHV-28, and blaOXA-1. Interestingly, blaCMY-2, the most common pAmpC β-lactamase genes reported worldwide, and mobile colistin resistance genes, mcr-10-1, were also identified. The presence of blaCMY-2 and mcr-10-1 is concerning as they may constitute a potentially high risk of pan-resistant post-surgical infections. It is imperative that healthcare professionals monitor intra-abdominal surgical site infections rigorously to prevent transmission of faecal ESBL carriage in high-risk patients.

Susceptibility of Gram-negative isolates collected in South Korea to imipenem/relebactam and comparator agents-SMART 2018-21.

To evaluate the in vitro susceptibility of recent Gram-negative pathogens collected in South Korean medical centres to imipenem/relebactam and comparator agents. From 2018 to 2021, six hospitals in South Korea each collected up to 250 consecutive, aerobic or facultative Gram-negative pathogens per year from patients with bloodstream, intra-abdominal, lower respiratory tract and urinary tract infections. MICs were determined using CLSI broth microdilution and interpreted by 2023 CLSI breakpoints. Most isolates that were imipenem/relebactam, imipenem or ceftolozane/tazobactam non-susceptible were screened for β-lactamase genes by PCR or WGS. Of all non-Morganellaceae Enterobacterales (NME) isolates (n = 4100), 98.8% were imipenem/relebactam susceptible. Most NME were also susceptible to imipenem alone (94.7%) and meropenem (97.3%); percent susceptible values for non-carbapenem β-lactam comparators were lower (68%-80%). Imipenem/relebactam retained activity against 96.4%, 70.8% and 70.6% of MDR, difficult-to-treat resistant (DTR) and meropenem-non-susceptible NME, respectively, and inhibited 93.1% of KPC-carrying and 95.5% of ESBL-carrying NME. Of imipenem/relebactam-resistant NME, 21/25 (84.0%) carried an MBL or an OXA-48-like carbapenemase. Of all Pseudomonas aeruginosa isolates (n = 738), 82.8% were imipenem/relebactam susceptible; percent susceptible values for all β-lactam comparators, including carbapenems (imipenem, meropenem) were 61.5%-74.7%. Less than 20% of MDR and DTR isolates, and 41% of meropenem-non-susceptible P. aeruginosa isolates were imipenem/relebactam susceptible. Of imipenem/relebactam-resistant P. aeruginosa isolates, 61.6% carried an MBL and 37.0% did not possess any acquired β-lactamase genes. Based on in vitro data, imipenem/relebactam, if licensed in South Korea, may be a viable treatment option for many hospitalized patients infected with common Gram-negative pathogens including NME exhibiting MDR, DTR and carbapenem resistance and many β-lactam-resistant phenotypes of P. aeruginosa.

In vitro activity of aztreonam-avibactam and comparators against Metallo-β-Lactamase-producing Enterobacterales from ATLAS Global Surveillance Program, 2016–2020

ObjectivesMetallo-β-lactamase (MBL)-producing Enterobacterales are a major challenge worldwide due to limited treatment options. Aztreonam-avibactam (ATM-AVI), which is under clinical development, has shown activity against MBL-positive isolates. This study evaluated the prevalence of MBL producers and the nature of enzymes among a global collection of clinical isolates of Enterobacterales from the Antimicrobial Testing Leadership and Surveillance program (ATLAS) surveillance program (2016–2020), and the antimicrobial activity of ATM-AVI and comparators against this collection. MethodsNon-duplicate clinical isolates of Enterobacterales (N = 106 686) collected across 63 countries were analysed. Antimicrobial susceptibility was performed using broth microdilution. Minimum inhibitory concentrations (MICs) were interpreted using Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing breakpoints. Provisional pharmacokinetic/pharmacodynamic breakpoint of ≤8 mg/L was considered for ATM-AVI. β-lactamase genes were characterized by polymerase chain reaction and sequencing. The Cochran Armitage Trend test was used to determine significant trends in percentage of isolates over time. ResultsOverall, MBL-positive isolates were 1.6% of total Enterobacterales isolates globally, with a significant increasing trend observed over time, globally and across regions (P < 0.05). New Delhi MBL (NDM) was the most common MBL (83.3%). ATM-AVI demonstrated potent activity against MBL-positive isolates (MIC ≤8 mg/L: 99.4% isolates inhibited; MIC90, 1 mg/L). Consistent activity was also noted across different regions. Potent activity was demonstrated against different NDM variants and MBL-positive isolates co-carrying other carbapenemases (98.1% and 99.7% isolates inhibited at ≤8 mg/L, respectively). About 0.6% MBL-positive isolates (10/1707) had MICs >8 mg/L for ATM-AVI. ConclusionATM-AVI demonstrated potent activity against MBL-positive isolates, including NDM variants and MBL-positive isolates co-carrying other carbapenemases, and may represent a good option for treating infections caused by MBL-positive Enterobacterales.

MOLECULAR ANALYSIS OF AMINOGLYCOSIDES AND β-LACTAMS RESISTANT GENES AMONG URINARY TRACT INFECTIONS

Urinary tract infection (UTI) is a multifactorial disease with a range of pathogenic microbes as causative agents including parasites, fungi and bacteria. However, the main causative pathogen in UTIs is bacteria. The UTI is one of the common reasons for nosocomial infection in the community. The current study aimed to determine the prevalence of UTIs followed by investigating the most effective antibiotics and the amplification of β-lactamase (bla-TEM) gene in isolated bacteria. The Present study was conducted in the tertiary care hospitals of various areas in Peshawar, Pakistan Mid-stream urine samples were collected from UTI patients. Inoculation of entire samples on MacConkey and Blood agar followed by urease, catalase test for gram-positive and citrate, and indole test for gram-negative bacteria was done. Culture sensitivity analysis for various antibiotics was done according to CLSI guidelines. DNA extraction and molecular characterization of bla-TEM resistance genes was also performed using specific primers by Polymerase Chain Reaction (PCR) technique. 117/531 (22.03%) samples were positive for UTI. Among these, most of the samples belong to female population 64.9%. In the age-wise distribution, the middle to older age group has the highest prevalence of UTI. 117 isolates separated with E. coli (62.4%) followed by Klebsiella spp (11.1%) and 7.7% of Pseudomonas as the most prevalent urinary tract infectious microbes. Antibiotic susceptibility showed gentamicin and meropenem as the most resistive antibiotics. Meropenem and ciprofloxacin were the most sensitive antibiotics. Molecular characterization showed 69/117 (58.9%) and 61/117 (52.1%) sample amplification for β-lactamase and aminoglycoside-resistant genes, respectively. It is concluded that UTI is a serious multi-pathogenic problem prevailing in District Peshawar. Most of the uropathogens show resistance towards different antibiotics based on the resistance genes detected in them.

Extensively drug-, ciprofloxacin-, cefotaxime-, and azithromycin-resistant Salmonella enterica serovars isolated from camel meat in Egypt

Given the great importance of Salmonella as a leading foodborne pathogen of global concern and the few available data regarding its prevalence in camel meat, the present study aimed to determine the prevalence, antimicrobial resistance (AMR) profile, virulence genes, β-lactamase genes of Salmonella enterica serovars isolated from camel meat marketed in Egypt. Forty-five (29.6 %) of the 152 camel meat samples examined were positive for Salmonella spp. Among the 432 Salmonella presumptive colonies isolated, 128 were molecularly verified as Salmonella after confirmation by PCR targeting the Salmonella marker (invA) gene. Virulence genes, encompassing stn, spvC, and hilA genes, were detected in 91.4 % (117/128), 20.3 % (26/128), and 80.5 % (103/128) of the isolates, respectively. S. Enteritidis, S. Typhimurium, S. Cerro, and S. Montevideo were the most prevalent serovars with incidences of 25 % (32/128), 15.6 % (20/128), 15.6 % (20/128), and 12.5 % (16/128), respectively. Interestingly, 56.3 %, 53.1 %, 37.5 %, 28.1 %, 21.9 %, 18.8 %, 12.5 %, and 3.1 % of the isolates tested showed resistance to cefepime, ciprofloxacin, levofloxacin, cefotaxime, gentamicin, colistin, meropenem, and azithromycin, respectively. Salmonella isolates showed resistance to at least one antibiotic, with a mean multiple antibiotic resistance (MAR) index of 0.472. Interestingly, 59.4 %, 15.6 %, and 3.1 % of the isolates were categorized into multidrug-resistant, extensively drug-resistant, and pan-drug-resistant, respectively. Only 23 (25 %) of the 92 ampicillin-resistant isolates were proven to be Extended Spectrum Beta-Lactamase (ESBL)-producing Salmonella, in which β-lactamase (bla) genes were detected. The blaOXA-2 was the most existing gene where it was detected lonely in 10 of the ampicillin-resistant isolates and coexisted with blaCMY-1 in 4 isolates and with blaCMY-2 in a single isolate. The blaCMY-1, however, existed in 11 isolates, whereas the blaCMY-2 gene was only detected in 3 isolates tested. The present findings affirm that camel meat could be a leading reservoir for multi- and extensively-drug-resistance β-lactamase-producing Salmonella, representing a global public health challenge. Therefore, further research is necessary to detect the prevalence and AMR of Salmonella serovars from camel meat in Egypt and other countries to put camel meat as a source of Salmonella in foods of animal origin.

Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of β-Lactamase Genes and Their RNA Transcripts.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to β-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.

Genomic Profiling of Multidrug-Resistant Swine Escherichia coli and Clonal Relationship to Human Isolates in Peru.

The misuse of antibiotics is accelerating antimicrobial resistance (AMR) in Escherichia coli isolated from farm animals. The genomes of ten multidrug-resistant (MDR) E. coli isolates from pigs were analyzed to determine their sequence types, serotypes, virulence, and AMR genes (ARGs). Additionally, the relationship was evaluated adding all the available genomes of Peruvian E. coli from humans using the cgMLST + HierCC scheme. Two aEPEC O186:H11-ST29 were identified, of which H11 and ST29 are reported in aEPEC isolates from different sources. An isolate ETEC-O149:H10-ST100 was identified, considered a high-risk clone that is frequently reported in different countries as a cause of diarrhea in piglets. One ExPEC O101:H11-ST167 was identified, for which ST167 is an international high-risk clone related to urinary infections in humans. We identified many ARGs, including extended-spectrum β-lactamase genes, and one ETEC harboring the mcr-1 gene. CgMLST + HierCC analysis differentiated three clusters, and in two, the human isolates were grouped with those of swine in the same cluster. We observed that Peruvian swine MDR E. coli cluster with Peruvian E. coli isolates from healthy humans and from clinical cases, which is of great public health concern and evidence that AMR surveillance should be strengthened based on the One Health approach.

Antimicrobial Resistance Profiles of Multidrug-Resistant Enterobacteria Isolated from Feces of Weaned Piglets.

Nosocomial infections caused by multidrug-resistant enterobacteria have become a major challenge in global public health. Previous studies have indicated that use of antibiotics in livestock production chains is linked to the rising threat of antibiotic resistance in humans. In this study, we aimed to evaluate the distribution of genes encoding resistance to tetracycline, β-lactams, and colistin in multidrug-resistant enterobacteria isolated from feces of weaned pigs. Ninety-four enterobacteria isolates were submitted to antibiotic susceptibility test by minimum inhibitory concentration (MIC). In addition, we performed conjugation experiments to verify if plasmid-bearing isolates containing the mcr-1 gene could transfer their resistance determinant to a colistin-sensitive recipient strain. Our results demonstrated a positive association between the detection of antibiotic resistance genes in enterobacteria and the phenotypic resistance profiles of the bacterial isolates. At least one of the extended-spectrum β-lactamases (ESBL) genes (blaCTX-M, blaTEM, or bla SHV) and tetA was found among most bacterial genera analyzed. In addition, results revealed that the mcr-1 gene can be transferred from E. coli UFV-627 isolate to an F- recipient (Escherichia coli K12) by conjugation. Our findings support the hypothesis that swine represents an important reservoir of antibiotic resistance genes and suggest that horizontal transfer mechanisms (e.g., conjugation) may mediate the spread of these genes in the swine gastrointestinal tract.

Effect of Nk-lysin peptides on bacterial growth, MIC, antimicrobial resistance, and viral activities

NK-lysins from chicken, bovine and human are used as antiviral and antibacterial agents. Gram-negative and gram-positive microorganisms, including Streptococcus pyogenes, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Shigella sonnei, Klebsiella pneumoniae and Salmonella typhimurium, are susceptible to NK-lysin treatment. The presence of dominant TEM-1 gene was noted in all untreated and treated bacteria, while TOHO-1 gene was absent in all bacteria. Importantly, β-lactamase genes CTX-M-1, CTX-M-8, and CTX-M-9 genes were detected in untreated bacterial strains; however, none of these were found in any bacterial strains following treatment with NK-lysin peptides. NK-lysin peptides are also used to test for inhibition of infectivity, which ranged from 50 to 90% depending on NK-lysin species. Chicken, bo vine and human NK-lysin peptides are demonstrated herein to have antibacterial activity and antiviral activity against Rotavirus (strain SA-11). On the basis of the comparison between these peptides, potent antiviral activity of bovine NK-lysin against Rotavirus (strain SA-11) is particularly evident, inhibiting infection by up to 90%. However, growth was also significantly inhibited by chicken and human NK-lysin peptides, restricted by 80 and 50%, respectively. This study provided a novel treatment using NK-lysin peptides to inhibit expression of β-lactamase genes in β-lactam antibiotic-resistant bacterial infections.

Mobile class A β-lactamase gene bla GMA-1.

Despite increasing reports, class A β-lactamases of environmental bacteria remain very poorly characterized, with limited understanding of their transmission patterns. To address this knowledge gap, we focused on a recently designated GMA family β-lactamase gene, bla GMA-1, found in marine bacterial genera such as Vibrio. This study shows that gammaproteobacterial mobile class A β-lactamase is specialized for penicillin degradation, and bla GMA-1 is frequently linked to strand-biased circularizing integrative elements (SEs) in sequences in the RefSeq/GenBank database. Evidence for the implication of SEs in β-lactamase environmental transmission provides insights for future surveillance studies of antimicrobial resistance genes in human clinical settings.