Β-lactamase Production Research Articles

Enterobacteriaceae isolates from clinical and household tap water samples: antibiotic resistance, screening for extended-spectrum, metallo- and ampC-beta-lactamases, and detection of blaTEM,blaSHV and blaCTX-M in Uyo, Nigeria.

Infections caused by multidrug-resistant (MDR) bacteria, extended spectrum β-lactamase (ESBL), metallo-β-lactamase (MBL) and AmpC-β-lactamase (AmpC-βL)-producers are increasing globally. This study identified bacteria in clinical and tap water samples and determined the prevalence of MDR, and β-lactamase enzymes and genes. Isolates were identified by the Vitek 2 (bioMérieux, France) automated system. Antibiotic resistance and screening for β-lactamase enzymes and genes was done using disc diffusion method and Vitek 2 automated system, CHROMagar-ESBL, combined double disc, inhibition-based method and multiplex polymerase chain reaction, respectively. The Enterobacteriaceae isolates obtained were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Salmonella spp., Proteus mirabilis, Enterobacter aerogenes, Shigella sonnei, Proteus vulgaris, Enterobacter sakazakii, Klebsiella oxytoca, Citrobacter diversus, and Serratia liquefaciens. Of the 674 isolates from clinical samples, 36.5%, 28.5%, and 19.9% were ESBL, MBL, and AmpC-βL producers, respectively. A low prevalence of AmpC-βL and MBL producers were obtained, with no significant difference (p<0.05) between the prevalence of ESBL and non-ESBL producers. Isolates exhibited varied levels of resistance to gentamicin, amoxicillin-clavulanic acid, ciprofloxacin, and tetracycline. The results showed that 54.6% of ESBL producers, 57.9% of MBL producers, and 62.8% of AmpC-βL producers were MDR strains. Of the 141 representative isolates tested, 36.9%, 15.6%, and 20.6% had only blaTEM, blaSHV, and blaCTX-M, respectively; 5.7% possessed both blaTEM and blaSHV; 7.1% possessed both blaTEM and blaCTX-M and 4.3% had both blaSHV and blaCTX-M. This study found a high prevalence of β-lactamase producers, indicating the need for further research on the molecular epidemiology of β-lactamase producers and their impacts in the region.

High Fecal Carriage of Extended-Spectrum β-Lactamase Producing Enterobacteriaceae by Children Admitted to the Pediatric University Hospital Complex in Bangui, Central African Republic

Antimicrobial resistance (AMR) is a global public health threat. Quality data on AMR are needed to tackle the rise of multidrug-resistant clones. These data are rare in low-income countries, especially in sub-Saharan Africa. In this study, we investigated the rise of extended-spectrum β-lactamase–producing (ESBL) Enterobacteriaceae in Bangui, Central African Republic. We collected 278 fecal samples from 0–5-year-old children admitted to the Pediatric University Hospital Complex in Bangui from July to September 2021. Enterobacteriaceae were isolated and identified, and their susceptibility to 19 antibiotics was tested. We recovered one and two Enterobacteriaceae species from 208 and 29 samples, respectively. One clone of each species from each sample was further characterized, for a total of 266 isolates. Escherichia coli predominated, followed by Klebsiella. AMR was frequent, with 98.5% (262/266) of the isolates resistant to at least one antibiotic. Additionally, 89.5% (238/266) of the isolates were multidrug resistant, with resistance being frequent against all tested antibiotics except carbapenems and tigecycline, for which no resistance was found. Importantly, 71.2% (198/278) of the children carried at least one ESBL species, and 85.3% (227/266) of the isolates displayed this phenotype. This study confirms the rise of ESBL Enterobacteriaceae in Bangui and stresses the need for action to preserve the efficacy of antibiotics, as crucial for the treatment of bacterial infections.

Emergence of colistin resistance and characterization of antimicrobial resistance and virulence factors of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from hybrid red tilapia cage culture.

Tilapia is a primary aquaculture fish in Thailand, but little is known about the occurrence of antimicrobial resistance (AMR) in Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae colonizing healthy tilapia intended for human consumption and the co-occurrence of these AMR bacteria in the cultivation water. This study determined the phenotype and genotype of AMR, extended-spectrum β-lactamase (ESBL) production, and virulence factors of A. hydrophila, Salmonella spp., and V. cholerae isolated from hybrid red tilapia and cultivation water in Thailand. Standard culture methods such as USFDA's BAM or ISO procedures were used for the original isolation, with all isolates confirmed by biochemical tests, serotyping, and species-specific gene detection based on PCR. A total of 278 isolates consisting of 15 A. hydrophila, 188 Salmonella spp., and 75 V. cholerae isolates were retrieved from a previous study. All isolates of A. hydrophila and Salmonella isolates were resistance to at least one antimicrobial, with 26.7% and 72.3% of the isolates being multidrug resistant (MDR), respectively. All A. hydrophila isolates were resistant to ampicillin (100%), followed by oxytetracycline (26.7%), tetracycline (26.7%), trimethoprim (26.7%), and oxolinic acid (20.0%). The predominant resistance genes in A. hydrophila were mcr-3 (20.0%), followed by 13.3% of isolates having floR, qnrS, sul1, sul2, and dfrA1. Salmonella isolates also exhibited a high prevalence of resistance to ampicillin (79.3%), oxolinic acid (75.5%), oxytetracycline (71.8%), chloramphenicol (62.8%), and florfenicol (55.3%). The most common resistance genes in these Salmonella isolates were qnrS (65.4%), tetA (64.9%), bla TEM (63.8%), and floR (55.9%). All V. cholerae isolates were susceptible to all antimicrobials tested, while the most common resistance gene was sul1 (12.0%). One isolate of A. hydrophila was positive for int1, while all isolates of Salmonella and V. cholerae isolates were negative for integrons and int SXT. None of the bacterial isolates in this study were producing ESBL. The occurrence of mcr-3 (20.0%) in these isolates from tilapia aquaculture may signify a serious occupational and consumer health risk given that colistin is a last resort antimicrobial for treatment of Gram-negative bacteria infections. Findings from this study on AMR bacteria in hybrid red tilapia suggest that aquaculture as practiced in Thailand can select for ubiquitous AMR pathogens, mobile genetic elements, and an emerging reservoir of mcr and colistin-resistant bacteria. Resistant and pathogenic bacteria, such as resistance to ampicillin and tetracycline, or MDR Salmonella circulating in aquaculture, together highlight the public health concerns and foodborne risks of zoonotic pathogens in humans from cultured freshwater fish.

Development of a Method for the Fast Detection of Extended-Spectrum β-Lactamase- and Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae from Dogs and Cats in the USA.

Antibiotic resistance, such as resistance to beta-lactams and the development of resistance mechanisms, is associated with multifactorial phenomena and not only with the use of third-generation cephalosporins. Many methods have been recommended for the detection of ESBL and pAmpC β-lactamase production but they are very subjective and the appropriate facilities are not available in most laboratories, especially not in clinics. Therefore, for fast clinical antimicrobial selection, we need to rapidly detect ESBL- and pAmpC β-lactamase-producing bacteria using a simple method with samples containing large amounts of bacteria. For the detection of ESBL- and pAmpC phenotypes and genes, the disk diffusion test, DDST and multiplex PCR were conducted. Of the 109 samples, 99 (90.8%) samples were grown in MacConkey broth containing cephalothin, and 71 samples were grown on MacConkey agar containing ceftiofur. Of the 71 samples grown on MacConkey agar containing ceftiofur, 58 Escherichia coli and 19 Klebsiella pneumoniae isolates, in particular, harbored β-lactamase genes. Of the 38 samples that did not grow in MacConkey broth containing cephalothin or on MacConkey agar containing ceftiofur, 32 isolates were identified as E. coli, and 10 isolates were identified as K. pneumoniae; β-lactamase genes were not detected in these E. coli and K. pneumoniae isolates. Of the 78 ESBL- and pAmpC β-lactamase-producing E. coli and K. pneumoniae, 55 (70.5%) isolates carried one or more ESBL genes and 56 (71.8%) isolates carried one or more pAmpC β-lactamase genes. Our method is a fast, and low-cost tool for the screening of frequently encountered ESBL- and pAmpC β-lactamase-producing bacteria and it would assist in diagnosis and improve therapeutic treatment in animal hospitals.

Bacteriological spectrum, extended-spectrum β-lactamase production and antimicrobial resistance pattern among patients with bloodstream infection in Addis Ababa

Bloodstream infection coupled with drug resistance in bloodborne bacteria is a major health problem globally. The current study sought to identify the bacterial spectrum, extended-spectrum -lactamase production, and antimicrobial resistance pattern in patients with bloodstream infection. This prospective cross-sectional study was conducted at Arsho Advanced Medical Laboratory, Addis Ababa, Ethiopia from January 2019- until July 2020. Blood collected from patients was inoculated into blood culture bottles and incubated appropriately. Identification, antimicrobial susceptibility testing, and extended-spectrum β-lactamase-production were determined with the VITEK 2 compact system. Of the samples collected, 156 (18.5%) were culture-positive. Klebsiella pneumoniae and Staphylococcus epidermidis were the dominant isolates. In Gram-negative bacteria, the prevalence of drug resistance was the highest against ampicillin (80.8%) and the lowest against imipenem (5.2%). While in Gram-positive bacteria it was the highest against clindamycin and the lowest against vancomycin and daptomycin. The prevalence of multi-drug resistance and extended-spectrum β-lactamase production of Gram-negative bacteria were 41.6% and 34.2%, respectively. The prevalence of bloodstream infection was 18.5%. Serious life-threatening pathogens including S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter spp was predominant. The prevalence of multi-drug resistance to both Gram-positive and Gram-negative bacteria and extended-spectrum β-lactamase-production were high but prevalence of carbapenem resistance was low. All these situations call for the establishment of strong infection control strategies, a drug regulatory system, and established antibiotic stewardship in healthcare settings.

House Flies (Musca domestica) from Swine and Poultry Farms Carrying Antimicrobial Resistant Enterobacteriaceae and Salmonella.

The house fly (Musca domestica) is a very common insect, abundantly present in farm settings. These insects are attracted by organic substrates and can easily be contaminated by several pathogenic and nonpathogenic bacteria. The aim of this survey was to evaluate the presence of Salmonella spp. and other Enterobacteriaceae in house flies captured in small-medium size farms, located in Northwest Tuscany, Central Italy, and to evaluate their antimicrobial resistance; furthermore, isolates were tested for extended spectrum β-lactamase and carbapenems resistance, considering the importance these antimicrobials have in human therapy. A total of 35 traps were placed in seven poultry and 15 swine farms; three different kinds of samples were analyzed from each trap, representing attractant substrate, insect body surface, and insect whole bodies. Enterobacteriaceae were isolated from 86.36% of farms, 82.87% of traps, and 60.95% of samples; high levels of resistance were detected for ampicillin (61.25% of resistant isolates) and tetracycline (42.5% of resistant isolates). One extended spectrum β-lactamase producer strain was isolated, carrying the blaTEM-1 gene. Salmonella spp. was detected in 36.36% of farms, 25.71% of traps, and 15.24% of samples. Five different serovars were identified: Kentucky, Kisarawe, London, Napoli, and Rubislaw; some isolates were in R phase. Resistance was detected mainly for ampicillin (31.21%) and tetracycline (31.21%). House flies could represent a serious hazard for biosecurity plans at the farm level, carrying and sharing relevant pathogenic and antimicrobial resistant bacteria.

First Report Occurrence of CIT and DHA AmpC β-lactamase Gene in Escherichia coli and Klebsiella pnuemoniae from Clinical Sample in South Eastern, Nigeria

Background and Objectives: Over time, the enzymes AmpC β-lactamases have become more significant, due to their roles in antibiotic resistance among enterobacteriaceace especially in Escherichia coli and Klebsiella pnuemoniae. Due to increase multidrug resistant express by AmpC β-lactamases producing bacteria strain, the patients care in several hospital has been severely hampered. Hence, this study was designed to assess the occurrence of CIT and DHA AmpC β-lactamase gene in Escherichia coli and Klebsiella pnuemoniae from clinical sample in south eastern, Nigeria&#x0D; Methodology: This study was conducted over an 8-month period on sixteen (16) non-repetitive clinical isolates of Escherichia coli and Klebsiella pnuemoniae collected from medical microbiology laboratory unit of Alex Ekweume Federal University Teaching Hospital in Abakaliki, Nigeria. The isolates were further identified using Standard microbiological Techniques and screened for cefoxitin resistance using a disc diffusion assay, followed by phenotypic tests using phenyl boronic acid assays for confirmation of AmpC β-lactamases production. Escherichia coli and Klebsiella pneumoniae strains were further screen for AmpC β-lactamase CIT and DHA genotype by polymerase chain reactions&#x0D; Result: Of the sixteen (16) confirmed phenotypic AmpC β-lactamase producing bacteria, 100% of the AmpC β-lactamase genes (DHA and CIT) were detected in E. coli from wound and urine samples from both male and female patients. The overall proportion of AmpC β-lactamases gene in Klebsiella pneumoniae were DHA (100 %) and CIT (100 %), in both male and female.&#x0D; Conclusion: This study indicate the occurrence of CIT and DHA AmpC genotype. The detection of AmpC β-lactamases in this study is of clinically importance as such bacteria are often MDR. Thus, being aware of the presence of AmpC β-lactamase-producing bacteria could be very beneficial for achieving more accurate epidemiological results as well as controlling their spread, while surveillance is required to track any further dissemination and emergence of other AmpC β-lactamase genotypes.

An Arsenal of Multiple Antimicrobial Resistance, Toxins, and Virulence Factors in Gram-Negative Bacterial Isolates from Food - A Formidable Combination!

Infectious diseases caused by pathogenic members of the family Enterobacteriaceae cause mortality and morbidity in humans. These are mediated mainly via toxins or virulence factors in combination with multiple antimicrobial resistance (MAR) against antimicrobials intended to treat infections. Resistance can be transferred to other bacteria, possibly also in association with other resistance determinants and/or virulence properties. Food-borne bacterial infections are one of the major causes of infections in humans. The level of scientific information about foodborne bacterial infections in Ethiopia is very limited at best. Bacteria were isolated from commercial dairy foods. These were cultured in appropriate media for identification at the family level (Enterobacteriaceae) based on Gram-negative, catalase-positive, oxidase-negative, and urease-negative phenotypes, followed by testing for the presence of virulence factors and resistance determinants to various antimicrobial classes using phenotypic and molecular tests. Twenty Gram-negative bacteria isolated from the foods were found to be resistant to almost all antimicrobials belonging to the phenicol, aminoglycoside, fluoroquinolone, monobactam, and β-lactam classes. All of them were multiple-drug-resistant. The resistance to the β-lactams was due to the production of β-lactamases and were also mostly resistant to some of the β-lactam/β-lactamase inhibitor combinations. Some isolates also contained toxins. This small-scale study demonstrated the presence, in the isolates, of high levels of virulence factors and resistance to major antimicrobials that are in clinical use. Most treatment being empirical, there can be not only a high degree of treatment failure but also the likelihood for further development and dissemination of antimicrobial resistance. Since dairy foods are animal products, there is an urgent need to control animal-food-human transmission mechanisms, restrict antimicrobial use in animal agriculture, and improve clinical treatment from the usual empirical treatment to more targeted and effective treatment.

Extensively Drug-Resistant Typhoid: Antibiotic Susceptibility Pattern of an Emerging Menace in Karachi

Background: Salmonella typhi, a rod-shaped Gram-negative bacterium that stands responsible for causing typhoid fever, in places with poor sanitation and polluted water. Injudicious antibiotic practices have significantly added to the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Salmonella typhi. This study aimed to determine the frequency and antibiotic susceptibility pattern of Salmonella typhi in blood cultures of patients with fever. Methods: This cross-sectional retrospective study was conducted from November 2018 to November 2019 at Tahir Laboratory, Karachi. All clinically suspected typhoid fever cases were cultured and Salmonella typhi was identified by traditional methods using standard microbiological procedures. Antibiotic susceptibility testing was done by the Kirby-Bauer disk diffusion method and a combined disc test was done to detect Extended Spectrum β-Lactamase production in XDR cases. Results: Out of 394 samples received for blood culture, 99(25.12%) were gram-positive, 158(40.10%) were gram-negative and 137 (34.7%) showed no growth. In 158 gram-negative samples, 92(58.22%) were of Salmonella typhi, consisting of 40(43.47%) cases of children aged 0-10 years. MDR and XDR Salmonella typhi isolates were 25(27.2%) and 8(8.7%) respectively, showing a male preponderance of 20 (60.60%). Both the MDR and XDR strains were sensitive to Meropenem and Azithromycin. Extended Spectrum β-Lactamase production was confirmed by combined disc test in all the XDR cases. Conclusion: The emergence of multidrug-resistant and extensively drug-resistant Salmonella typhi requires surveillance to see antibiogram trends for empirical therapy and to facilitate the prevention of the spread of the disease. Keywords: Salmonella typhi; Antibiotic Susceptibility Test; Extensively Drug-Resistant; Multidrug-Resistant.

Phenotypic Detection of AmpC \(\beta\)-lactamase Producing Escherichia coli among Patients in Hospital Wards

Background and Objectives: AmpC β-lactamase producing Escherichia coli has emerged in hospital environments as an important health issue that is of a global health concern as drug-resistant pathogenic bacteria that has evolved into strains that are resistant to many classes of antibiotics. However, awareness of the prevalence of AmpC β-lactamase producing microorganisms such as E. coli could be very valuable for achieving more accurate epidemiological results, as well as controlling their spread in the different hospital wards.&#x0D; Methodology: A total of four hundred (400) clinical samples comprising of two hundred and seventy-nine (279) urine samples and one hundred and Twenty-one (121) wound samples were collected from patients in different ward at AE-FUTHA. The clinical samples were analyze using standard microbiological culture and identification of Escherichia coli. Detection of phenotypic AmpC β-lactamases production was performed using Cefoxitin-Cloxacillin Double-Disk Synergy Test (CC-DDST). Antibiotic susceptibility studies of AmpC β-lactamases producing Escherichia coli was determined using the Kirby–Bauer disk diffusion method and the results were construed using the Clinical Laboratory Standard Institute (CLSI) zone diameter breakpoints.&#x0D; Results: Phenotypic AmpC β-lactamases producing E. coli accounted overall prevalence rate of 94.1%. The frequency of AmpC β-lactamases producing E. coli in urine samples were 57(93.4%) comprising of high occurrence rate in Surgical ward 15(100%) followed by medical ward 24(88.9%) but also accounted for overall prevalence rate of 38(95.0%) in wound samples consisting of 9(100 %) from children ward and medical ward 10(100%) followed by Orthopedic ward 13(86.7%) with the least prevalence rate. The AmpC β-lactamases producing E. coli exhibited high percentage of resistance within the range of 50-100% against ceftriaxone, ceftazidime, cefepime, azetronam, Trimethoprim-Sulfamethoxazole Ticarcillin-clavulanic acid but were susceptible to ofloxacin 70.0%, Imipenem 83.3% and amikacin 100%.&#x0D; Conclusion: The findings of this study is a proof of the occurrence of AmpC β-lactamase producing Escherichia coli among patients admitted to hospital wards and there’s an urgent need for controlling and managing the development of MDR genotype strain. Moreover, to prevent the spread of resistance among AmpC β-lactamase producing Escherichia coli to other strains and improve the effectiveness of antibiotics, it is suggested to establish a precise schedule for antibiotic use in each region based on their antibiotic resistance pattern.

Molecular characterisation of a meropenem-resistant Haemophilus influenzae

Background: Haemophilus influenzae β-lactam resistance is mediated by production of β-lactamase and mutations in penicillin-binding protein 3 (PBP3, encoded by ftsI). Only one of the 646 patient-unique H. influenzae isolated in our laboratory between 2012–July 2022 was meropenem (amoxicillin, co-amoxiclav and ceftriaxone) resistant. Methods: Identification and susceptibility of this meropenem-resistant H. influenzae (MRHI) was confirmed by disk diffusion and E-test (bioMérieux) (CLSI M100). WGS was performed on GridION (Nanopore) and reference mapped with Geneious Prime 2022.2. PCR and Sanger sequencing was used to confirm results of WGS. Results: The MRHI isolate was β-lactamase negative and non-typeable. ftsI subgroup IIc1 mutations were present, signifying previously described low level PBP3 alterations that should not solely account for carbapenem resistance. Group VII2 acrR (repressor of AcrAB efflux pump) substitutions were observed but without the reported carbapenem resistance associated frameshift mutation. There were also multiple substitutions and insertions in ompP2 (outer membrane protein P2) porin, associated with carbapenem resistance.2 The 100% concordance between Sanger sequencing and Nanopore increases our confidence for application of this technology for future investigations. Conclusion: Meropenem resistance in this isolate is likely multifactorial, with PBP3 alterations and decreased influx as contributing factors.

Antibiotic Susceptibility and Molecular Typing of Invasive Haemophilus influenzae Isolates, with Emergence of Ciprofloxacin Resistance, 2017–2021, Italy

Haemophilus influenzae invasive disease is a severe infection that needs rapid antibiotic therapy. The aim of the study was to perform and evaluate the serotype distribution, antibiotic susceptibility and molecular characteristics of 392 H. influenzae invasive isolates collected during 2017-2021 in Italy. The majority of isolates were NTHi (305/392, 77.8%), followed by Hib (49/392, 12.5%). Ampicillin resistance was frequently detected (85/392, 21.7%): 12.2% were β-lactamase producers (all blaTEM except one blaROB), 9.4% were β-lactamase-negative ampicillin-resistant (BLNAR), with mutations in the ftsI gene. Six isolates were resistant to ciprofloxacin, with substitutions in GyrA and ParC. An MLST analysis revealed the occurrence of international resistant clones, such as ST103 and ST14, highlighting the importance of molecular surveillance.

High-dose Cefepime vs Carbapenems for Bacteremia Caused by Enterobacterales With Moderate to High Risk of Clinically Significant AmpC β-lactamase Production.

Limited data suggest that serious infections caused by Enterobacterales with a moderate to high risk of clinically significant AmpC production can be successfully treated with cefepime if the cefepime minimum inhibitory concentration (MIC) is ≤2 µg/mL. However, isolates with a cefepime-susceptible dose-dependent (SDD) MIC of 4-8 µg/mL should receive a carbapenem due to target attainment and extended-spectrum β-lactamase (ESBL) concerns. This was a retrospective cohort study of hospitalized patients with E. cloacae, K. aerogenes, or C. freundii bacteremia from January 2015 to March 2022 receiving high-dose cefepime or a carbapenem. Cox regression models were used with incorporation of inverse probability of treatment weighting and time-varying covariates. Of the 315 patients included, 169 received cefepime and 146 received a carbapenem (ertapenem n = 90, meropenem n = 56). Cefepime was not associated with an increased risk of 30-day mortality compared with carbapenem therapy (adjusted hazard ratio [aHR], 1.45; 95% CI, 0.79-2.14), which was consistent for patients with cefepime SDD isolates (aHR, 1.19; 95% CI, 0.52-1.77). Multivariable weighted Cox models identified Pitt bacteremia score >4 (aHR, 1.41; 95% CI, 1.04-1.92), deep infection (aHR, 2.27; 95% CI, 1.21-4.32), and ceftriaxone-resistant AmpC-E (aHR, 1.32; 95% CI, 1.03-1.59) to be independent predictors associated with increased mortality risk, while receipt of prolonged-infusion β-lactam was protective (aHR, 0.67; 95% CI, 0.40-0.89). Among patients with bacteremia caused by Enterobacterales with moderate to high risk of clinically significant AmpC production, these data demonstrate similar risk of 30-day mortality for high-dose cefepime or a carbapenem as definitive β-lactam therapy.

Staphylococcus aureus in Horses in Nigeria: Occurrence, Antimicrobial, Methicillin and Heavy Metal Resistance and Virulence Potentials

Staphylococcus aureus was isolated from a total of 360 nasal and groin skin swabs from 180 systematic randomly-selected horses slaughtered for meat at Obollo-Afor, Enugu State, Southeast Nigeria and antimicrobial, methicillin and heavy metal resistance profile and virulence potentials of the isolates established. Baird-Parker agar with egg yolk tellurite was used for S. aureus isolation. S. aureus isolates were confirmed biochemically and serologically using a specific S. aureus Staphytect Plus™ latex agglutination test kit. The antimicrobial resistance profile, methicillin, vancomycin and inducible clindamycin resistance, and β-lactamase production of the isolates were determined with disc diffusion. Tolerance to Copper, Cadmium, Lead and Zinc was assessed using the agar dilution method and virulence potentials were determined using phenotypic methods. Forty-three (23.9%) of the 180 horses harbored S. aureus. Some 71 S. aureus were recovered from the 360 samples. Two (2.8%) of the 71 S. aureus were methicillin-resistant S. aureus (MRSA) and 69 (97.2%) were methicillin-susceptible. MRSA was recovered from 2 (1.1%) of the 180 horses. Some 9.4% of the isolates were multiple drug-resistant (MDR). The mean multiple antibiotic resistance indices (MARI) for the isolates was 0.24. Heavy metal resistance rate of the isolates ranged between 35.4-70.4%. The isolates, including the MRSA strains, displayed virulence potentials as clumping factor and catalase, gelatinase, caseinase, heamolysin, and biofilm was at the rate of 100%, 53.5%, 43.7%, 18.3% and 23.9%, respectively. This study showed that a considerable percentage of horses slaughtered in Obollo-Afor Southeastern Nigeria are potential reservoirs of virulent multiple drug- and heavy metal-resistant S. aureus, including MRSA, that could spread to humans and the environment.

Performance of Phenotypic Tests to Detect β-Lactamases in a Population of β-Lactamase Coproducing Enterobacteriaceae Isolates.

Objectives This study aimed to evaluate the performance of routinely used phenotypic tests to detect β-lactamase production in isolates coproducing multiple β-lactamase types. Methods Commonly used phenotypic tests for the detection of extended spectrum β-lactamases (ESBL), AmpC β-lactamase, and carbapenemases were compared with detection and sequencing of β-lactamase genes (as the reference test) in 176 uropathogenic Enterobacteriaceae coproducing multiple β-lactamases from two hospitals in the Western Province of Sri Lanka. Results Majority of the isolates (147/176, 83.5%) carried β-lactamase genes with (90/147, 61%) harboring multiple genes. The Clinical and Laboratory Standards Institute screening method using cefotaxime (sensitivity [Se], 97; specificity [Sp], 93; accuracy [Ac], 94) and ceftriaxone (Se, 97; Sp, 91; Ac, 93) was the most effective to detect ESBLs. The modified double disc synergy test (Se, 98; Sp, 98; Ac, 97) and combined disc test (Se, 94; Sp, 98; Ac, 96) showed good specificity for confirmation of ESBLs. Cefoxitin resistance (Se, 97; Sp, 73; Ac, 85) and the AmpC disc test (Se, 96; Sp, 82; Ac, 86) were sensitive to detect AmpC β-lactamase producers coproducing other β-lactamases but showed low specificity, probably due to coproduction of carbapenemases. Meropenem was useful to screen for New Delhi metallo β-lactamases and OXA-48-like carbapenemases (Se, 97; Sp, 96; Ac, 96). The modified carbapenem inactivation method showed excellent performance (Se, 97; Sp, 98; Ac, 97) in identifying production of both types of carbapenemases and was able to distinguish this from carbapenem resistance due to potential mutations in the porin gene. Conclusion Microbiology laboratories that are still depend on phenotypic tests should utilize tests that are compatible with the types of β-lactamase prevalent in the region and those that are least affected by coexisting resistance mechanisms.

Colonization, Density, and Antibiotic Resistance of Streptococcus pneumoniae, Haemophilus Influenzae, and Moraxella catarrhalis among PCV13-Vaccinated Infants in the First Six Months of Life in Rochester, New York: A Cohort Study.

Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hflu), and Moraxella catarrhalis (Mcat) nasopharyngeal colonization precedes disease pathogenesis and varies among settings and countries. We sought to assess colonization prevalence, density, Spn serotypes, and antibiotic resistance in children in the first 6 months of life in pediatric primary care settings. Prospective cohort study in Rochester, NY during 2018-2020. Nasopharyngeal swabs were collected from 101 children at age 1, 2, and 3 weeks, then 1, 2, 4, 6, 9, 12, 15, 18, and 24 months. Spn serotypes were determined by Quellung. Oxacillin resistance for Spn and β-lactamase production by Hflu and Mcat was tested. All children received PCV13 vaccine according to U.S. recommended schedule. Spn, Hflu, and Mcat colonization was detected in only 5% of infants before age 2 months old. Cumulative prevalence was 34% for Spn, 10% for Hflu, and 53% for Mcat in children ≤6 months of age. Nasopharyngeal bacterial density of Spn, Hflu, and Mcat (x = 2.71 log) in children ≤6 months of age was lower than at 7-24 months of age (x = 3.15 log, p < 0.0001). Predominant serotypes detected ≤6 months of age were 23B (16.7%), 22F (12.9%), 15B/C (11%), and 16F (9.2%). In total, 14.8% of Spn isolates were oxacillin resistant and 66.7% of Hflu isolates were β-lactamase producing. Spn, Hflu, and Mcat nasopharyngeal colonization was uncommon and of low density among children ≤6 months old, especially among children <2 months of age. Non-PCV13 serotypes predominated and a different serotype distribution was observed in ≤6-month olds compared to 7- to 24-month olds.

Prevalence of Carbapenemase and Extended-Spectrum β-Lactamase Producing Enterobacteriaceae: A Cross-Sectional Study.

Enterobacteriaceae have been classified as severely drug resistant bacteria by the World Health Organization due to their extensive production and dissemination of carbapenemases (CPs) and extended-spectrum β-lactamases (ESBL). The current study was conducted with the aim to determine the prevalence of CP- and ESBL-producing Enterobacteriaceae, as well as their antibiotic susceptibility profiles. For this, a hospital-based study was conducted which included 384 participants with bacterial infections. The collection and processing of specimens was conducted per standard microbiological protocol. The samples were inoculated on agar media plates to obtain the bacterial growths, and if they were positive for any bacterial growth, the antibiotic susceptibility testing was performed using disk diffusion method to check their antibiotic susceptibility patterns. The double disc diffusion as well as carbapenem inhibition techniques were used to examine the CP enzymes. Multiplex real-time PCR technique was performed to identify three distinct genetic types of CPs that have been identified in the Enterobacteriaceae (KPC, NDM, and OXA-48). A majority of participants (58.3%) in the current study were living in urban areas. A total of 227 (59.1%) patients were hospitalized. Furthermore, 26.04% of the patients were determined to be suffering from infections with Enterobacteriaceae. Escherichia coli was the most prevalent (9.1%) isolate overall, followed by Klebsiella pneumoniae (8.07%), Acinetobacter baumannii (2.6%), Pseudomonas aeruginosa (3.1%), Enterobacter cloacae (1.3%), Proteus spp. (1.3%), and Morganella spp. (0.5%). The studied patients were suffering from urinary tract infections (48.6%), blood stream infections (32.2%), wounds infection (11.9%), and respiratory infections (7.03%), confirmed with bacterial cultures. The resistance against carbapenems was seen in 31.4% of E. coli isolates, 25.8% in K. pneumoniae, 50% in P. aeruginosa, 25% in A. baumannii, and 20% in E. cloacae isolates. Such high rates of CP- and ESBL-producing Enterobacteriaceae are alarming, suggesting high spread in the study area. It is advised to implement better infection prevention and control strategies and conduct further nationwide screening of the carriers of these pathogens. This might help in reducing the burden of highly resistant bugs.

Exploring broilers and native fowls of Andaman and Nicobar Islands as a source of β-lactamase-producing Enterobacteriaceae even with limited anthropogenic activities and docking-based identification of catalytic domains in novel β-lactamase variants.

The present study was conducted to detect the occurrence of β-lactamase and biofilm-producing Escherichia coli, Salmonella, and Klebsiella in broilers and native fowl reared in the Andaman and Nicobar Islands, India. The study also included molecular docking experiments to confirm the nature of the catalytic domains found in the β-lactamase variants obtained and to reveal the clonal relationship of the isolates with human clinical strains from the database. A total of 199 cloacal swabs were collected from five poultry breeds/varieties (broiler, Vanraja, Desi, Nicobari, and layer) in three districts of the Andaman and Nicobar Islands. E. coli, Salmonella enterica, and Klebsiella pneumoniae were isolated by standard techniques and confirmed by PCR. Phenotypical β-lactamase producers were identified by a double-disc test. The genes (bla CTX, bla SHV, bla TEM , and bla AmpC) were screened, and selected sequences of β-lactamase variants were submitted to DDBJ. Homology modeling, model validation, and active site identification of different β-lactamase variants were done by the SWISS-MODEL. Molecular docking was performed to identify the catalytic domains of the β-lactamase variants. The selected β-lactamase sequences were compared with the Indian ESBL sequences from human clinical strains in NCBI-GenBank. In total, 425 Enterobacteriaceae strains were isolated from the collected samples. Klebsiella pneumoniae (42.58%) was found to be the most prevalent, followed by Salmonella enterica (30.82%) and E. coli (26.58%). The phenotypical antibiogram of all 425 isolates showed the highest resistance against oxytetracycline (61-76%) and the lowest against gentamicin (15-20%). Phenotypical production of β-lactamase enzymes was observed in 141 (33.38%) isolates. The isolation rate of β-lactamase producing E. coli, Salmonella enterica, and Klebsiella pneumoniae was significantly higher (p < 0.05) in the birds reared in the South Andaman district (25.6, 17.5, and 18.7%, respectively) than in Nicobar (11.5, 7.6, 7.1%, respectively). Genotyping of the β-lactamase-producing isolates revealed the maximum possession of bla TEM, followed by bla SHV and bla CTX - M. The nucleotide sequences were found to be similar with bla CTX - M-15, bla SHV - 11, bla SHV - 27, bla SHV - 228, bla TEM - 1, and bla AmpC in BLAST search. Distribution of studied biofilm-associated genes in Enterobacteriaceae strains from different varieties of the birds revealed that the layer birds had the maximum possession, followed by Vanraja, Desi, broilers, and Nicobari fowls. The phylogenetic analysis of selected sequences revealed a partial clonal relationship with human clinical strains of the Indian subcontinent. Molecular docking depicted the Gibbs free energy release for 10 different macromolecules (proteins) and ligand (antibiotic) complexes, ranging from -8.1 (SHV-27 + cefotaxime) to -7 (TEM-1 + cefotaxime) kcal/mol. The study revealed β-lactamase variants circulating in the fowl population of the Andaman and Nicobar Islands (India), even in remote places with low anthropogenic activity. Most of the strains possessed bla TEM - 1, followed by bla CTX - M-15. Possession of bla SHV - 11, bla SHV - 27, and bla SHV - 228 in poultry Enterobacteriaceae strains was not reported earlier from any part of the world. The phylogenetic analysis revealed a partial clonal relationship of β-lactamase sequences with the human clinical strains isolated from the Indian subcontinent.

Antibacterial plant extracts inhibit the beta-lactamase of Escherichia coli clinical isolates

Beta-lactamase production is the main resistance mechanism to beta-lactam antibiotics by Enterobacteriaceae including Escherichia coli. In this study, we selected Terminalia superba, Annona senegalensis, Ipomoea batatas and Psidium guajava, which antibacterial properties have been proved with the aim to evaluate their beta-lactamase inhibitory activity. Production of β-lactamase was determined by modified tube acidimetric method using benzyl penicillin as substrate and phenol red in liquid medium. The tests were carried out on the ability of ethanolic plants extracts to reduce the hydrolysis of benzyl penicillin. Three plant extracts showed beta-lactamase inhibitory activity, with the highest enzyme inhibition rate found with Terminalia superba. Plant extracts with beta-lactamase inhibiting potential can be associated with beta-lactam antibiotics which are no longer effective to overcome the growing resistance to these widely prescribed antibiotics. The finding of this study showed the potential of antibacterial plants in the search for new beta-lactamase inhibitors.

Development of a whole-cell biosensor for β-lactamase inhibitor discovery.

The production of β-lactamases by bacterial pathogens endangers antimicrobial therapy, and new inhibitors for β-lactamases are urgently needed. We report the development of a luminescent-based biosensor that quantifies β-lactamase inhibition in a cellular context, based on the activation of transcriptional factor AmpR following the exposure of bacterial cells to β-lactams. This rapid method can account for factors like membrane permeability and can be employed to identify new β-lactamase inhibitors.