Β-lactamase Production Research Articles

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.

Multi-drug Resistance, β-Lactamases Production, and Coexistence of bla NDM-1 and mcr-1 in Escherichia coli Clinical Isolates From a Referral Hospital in Kathmandu, Nepal.

The ability of pathogenic Escherichia coli to produce carbapenemase enzymes is a characteristic that allows them to resist various antibiotics, including last-resort antibiotics like colistin and carbapenem. Our objectives were to identify rapidly developing antibiotic resistance (AR), assess β-lactamases production, and detect mcr-1 and bla NDM-1 genes in the isolates. A prospective cross-sectional study was carried out in a referral hospital located in Kathmandu from November 2019 to December 2020 using standard laboratory and molecular protocols. Among 77 total E. coli isolates, 64 (83.1%) of them were categorized as MDR. Phenotypically 13 (20.3%) colistin-resistant, 30 (46.9%) ESBL and 8 (12.5%) AmpC producers, and 5 (7.8%) ESBL/AmpC co-producers were distributed among MDR-E. coli. Minimum inhibitory concentrations (MIC) against the majority of MDR isolates were exhibited at 1 g/L. Of these 77 E. coli isolates, 24 (31.2%) were carbapenem-resistant. Among these carbapenem-resistant bacteria, 11 (45.9%) isolates were reported to be colistin-resistant, while 15 (62.5%) and 2 (8.3%) were MBL and KPC producers, respectively. Out of 15 MBL producers, 6 (40%) harbored bla NDM-1, and 8 (61.5%) out of 13 colistin-resistant pathogens possessed mcr-1. The resistance by colistin- and carbapenem were statistically associated (P < .001). However, only 2 (18.2%) of the co-resistant bacteria were found to have both genes. Our study revealed the highly prevalent MDR and the carbapenem-resistant E. coli and emphasized that the pathogens possess a wide range of capabilities to synthesize β-lactamases. These findings could assist to expand the understanding of AR in terms of enzyme production.

Characterization of Extended Spectrum β-lactamase and AmpC Producing Organisms Isolated from a Third Level Hospital Patients in Dominican Republic

Extended-spectrum β-lactamases (ESBLs) producing Enterobacteriaceae have become a challenge for clinicians as they frequently show co-resistance to other antibiotic classes. AmpC-type β-lactamases (AmpCs), tend to be more difficult to treat as they could be induced during antibiotic therapy. To characterize ESBLs and AmpCs producing organisms, we collected clinical samples identified through MicroScan® as members of the Enterobacteriaceae family and ESBLs producers, following Clinical Laboratory Standards Institute indications, from January through March 2021, in a third level hospital in Santiago de Los Caballeros, Dominican Republic. Samples underwent manual confirmation via ESBL + AmpC screen discs kit (Liofichelm® srl, Italy) and a genotype analysis was done by DNA extraction for detection of specific genes related with ESBL ((blaSHV, blaTEM, blaCTX, and blaCMY-4) or AmpC (CIT, MOX, DHA and FOX) expression. 54 samples were confirmed as ESBL and/or AmpC producers with Escherichia coli as the most frequent (33/61%) followed by Klebsiella sp. (9/17%). blaCTX was detected in 29 (67%) isolates, followed by blaTEM (23/53%) and blaSHV (18 /40%) . Among the AmpC encoding genes, DHA and CIT gene pools were detected in 3 (7%) and 2 (5%) of the samples, respectively. Two or more genes were detected in 19 (44%) samples. All the AmpC genes were found in an isolate which already had, at least, two other β-lactamase encoding genes. In this cohort, ESBLs and AmpCs -producing organisms with multiple resistance genes were detected. Efforts to curb antibiotic availability and to establish antimicrobial stewardship programs are needed.

Hospital-acquired infections due to carbapenem-resistant Providencia stuartii.

During the course of a retrospective survey on healthcare associated infections (HAIs) due to carbapenem-resistant organisms, an unusual prevalence of HAIs due to carbapenem-resistant Providencia stuartii (CRPS) was found. Hence this study aimed to conduct the occurrence of P. stuartii associated HAIs with special reference to the drug resistance profiling of these isolates. Of the eight total HAI cases (7.5% of total HAIs and 33.3% of HAIs due to Enterobacterales) of CRPS infections included in this study, three were reported from ventilator-associated pneumonia (VAP), three were surgical site infections (SSIs), one was a catheter-associated urinary tract infection (CAUTI) and one was a bloodstream infection. All the eight CRPS isolates were tested for extended-spectrum β-lactamases production, AmpC hyperproduction as well as carbapenem resistance. Typing of the isolates was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR). All the eight isolates of CRPS were found to be AmpC hyperproducers, carbapenemase producers, and harboured chromosomally located blaNDM in seven isolates and blaIMP genes in one. All the cases with CRPS infections had prior history of colistin therapy along with prolonged hospital stay (>20 days). The cases were located in five different wards/intensive care unit (ICU) within the hospital in one year. However, strain typing by REP-PCR revealed 100 per cent similarity and clonal relatedness in all the seven isolates carrying blaNDM genes. Interestingly, routine hospital surveillance revealed a high carriage of P. stuartii in the axilla of patients admitted to the ICU. The study findings suggest CRPS as an important cause of HAIs. This organism often goes unnoticed due to the burden of carbapenem resistance in other Enterobacterales and non-fermenters.

BETA-LACTAMASES: PHENOTYPIC IDENTIFICATION METHODS

Today, β-lactam antibiotics are the most widely used. This is due to their low toxicity, wide spectrum of action and variety of drugs. Therefore, the bacterial resistance factor in the form of β-lactamase production is of particular interest. Bacteriological laboratories have classical methods for detecting β-lactamases. However, the combination of bacterial production of enzymes of different classes, together with the overexpression of some of them, causes difficulties in recognizing the production of extended-spectrum enzymes, which is critical for making clinical decisions. Currently known phenotypic detection methods for β-lactamases and extended-spectrum carbapenemases, when performed correctly, allow precise elucidation of the mechanism of resistance. However, these methods are very labor-intensive, difficult to implement and require highly qualified staff of bacteriological laboratories. Perhaps methods based on molecular diagnostic approaches, due to automation and standardization, high accuracy and specificity in identifying β-lactamase genes, reducing research time, and the ability to identify uncultivable or slowly growing microorganisms, could simplify the determination of mechanisms of antibiotic resistance in the future.

Characterization of extended spectrum β-lactamase producing Escherichia coli strains isolated from urogenital system of dogs in Van province of Turkey.

Escherichia coli is a bacterial agent that causes urogenital system infection in dogs. Beta-lactam (β-lactam) group antibiotics are frequently used in the treatment of E. coli infections. This study aimed to investigate the presence of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC in E. coli strains isolated from the urogenital tracts of 125 dogs. Fifty E. coli strains were identified by conventional bacteriological and PCR methods. Disk diffusion method was used for the determination of antimicrobial susceptibility of the isolates as well as productions of plasmidic AmpC and ESBL. The presence of blaTEM, blaSHV, and blaCTX-M group genes was determined in the isolates by PCR. ERIC-PCR was also used for genotyping of the isolates. Although 22 (44%) of 50 E. coli isolates were found to be ESBL positive, no isolate shows plasmidic AmpC β-lactamase production. Among 22 ESBL positive isolates, blaTEM, blaSHV, and blaCTX-M group 1 genes were found in 11 (50%), 1 (4.54%), and 6 (27.27%) isolates, respectively. The highest resistance was observed against tetracycline (28%), followed by streptomycin (24%), trimethoprim-sulfamethoxazole (24%), and chloramphenicol (22%), respectively. In the isolates, 11 different main profiles were also determined by ERIC-PCR. It was shown that ESBL positive isolates were related to G10 profiles. The use of extended spectrum β-lactam group antibiotics for the treatment of E. coli infections in dogs is critical; nevertheless, they may not be effective due to the high rate of resistance to this antibiotic group in E. coli.

Multidrug resistant and extended spectrum β-lactamase producing gram negative bacterial uropathogens among females in a tertiary hospital, Pokhara

Introduction: Extended spectrum β-lactamase producing gram negative bacilli has emerged and spread worldwide as an important cause of urinary tract infections and indicates a major threat. The study aimed to determine the prevalence of multidrug resistant and extended spectrum beta-lactamase producing gram negative bacterial uropathogens among females in a tertiary level hospital. Methods: A hospital-based cross-sectional study was conducted in Pokhara Academy of Health Sciences, Pokhara from February to November, 2019. A total number of 301 midstream urine samples were collected and examined using MacConkey agar and blood agar medium. Antimicrobial susceptibility testing was done by Kirby Bauer disk diffusion method on Mueller Hinton agar using Clinical and Laboratory Standards Institute guidelines. Results: Out of 301 mid-stream urine samples, 99(33%) sample showed significant bacterial growth. Among them, 78(79%) were gram negative bacteria. Escherichia coli were the predominant organism. Multidrug resistant gram negative isolates were 65.4%. Among 78 Gram negative isolates, 31(39.7%) were extended spectrum β-lactamase producers. Among extended spectrum β-lactamase producers, 27(87.1%) were MDR. Highest frequency of extended spectrum β-lactamases production was seen in E. coli, 23(74.2%). Majority of gram negative bacteria showed susceptibility toward colistin and nitrofurantoin. Ampicillin was found to be highly resistant towards gram negative uropathogen. Conclusions: This study found that higher proportion of multi-drug resistants were among gram negative isolates and further more among extended spectrum β-lactamase producing gram negative isolates. Thus, there is urgent need to address the issue of antimicrobial resistant and promote rational use of the antibiotics in our region.

In vitro Activity of Ceftaroline Against an International Collection of Kingella kingae Isolates Recovered From Carriers and Invasive Infections.

Improvements in blood culture techniques and molecular-based diagnostics have led to increased recognition of Kingella kingae as an invasive human pathogen causing bacteremia, septic arthritis, osteomyelitis and endocarditis in young children. Serious disease and potentially life-threatening complications of infection due to K. kingae necessitate timely identification and appropriate antimicrobial therapy. Ceftaroline is a fifth-generation broad spectrum cephalosporin that possesses activity against Gram-negative and Gram-positive pathogens similar to third-generation cephalosporins, but also includes methicillin-resistant Staphylococcus aureus . This study reports the in vitro activity of ceftaroline and comparator agents against an international collection of K. kingae isolates. A collection of 308 K. kingae isolates was obtained primarily from children with bacteremia, endocarditis, osteoarticular infections or from asymptomatic pediatric carriers. Isolates were tested for antibiotic susceptibility using Clinical and Laboratory Standard Institute broth microdilution methodology and screened for β-lactamase production using a nitrocefin chromogenic test. Ceftaroline inhibited all K. kingae isolates at ≤0.06 mg/L (MIC 50/90 , 0.015/0.03 mg/L). Ceftaroline MICs were similar to results with ceftriaxone (MIC 50/90 , 0.015/0.015 mg/L), meropenem (MIC 50/90 , 0.015/0.015 mg/L) and ampicillin-sulbactam (MIC 50/90 , 0.06/0.06 mg/L). Ceftaroline MICs were slightly lower than MICs for cefuroxime and amoxicillin/clavulanate (MIC 50/90 , 0.06/0.12 mg/L). MICs were high for clindamycin (MIC 50/90 , 2/4 mg/L) and oxacillin (MIC 50/90 , 4/8 mg/L). Sixteen isolates (5.2%) yielded a positive nitrocefin test indicating production of β-lactamase; ceftaroline demonstrated equivalent MICs against β-lactamase - positive and β-lactamase - negative strains (MIC 50/90 , 0.015/0.3 mg/L). The potent activity of ceftaroline against this large international collection of K. kingae isolates supports further clinical evaluation in children.

Identification of cfxA gene variants and susceptibility patterns in β-lactamase-producing Prevotella strains

ObjectivesAntimicrobial-resistant isolates of Prevotella species, especially those resistant to β-lactams, have become increasingly common. Here, we aimed to elucidate the underlying mechanisms contributing to the emergence and spread of antimicrobial resistance in Prevotella species. MethodsPrevotella species were isolated from a variety of clinical specimens. β-lactamase production was determined using nitrocefin discs, and the determination of minimum inhibitory concentration (MIC) to ten antimicrobials was done by the agar dilution method. Four resistance genes (cfxA, tetQ, ermF, and nim) and cfxA-flanking regions were detected using polymerase chain reaction. cfxA and the flanking regions were sequenced, and a phylogenetic tree was constructed based on CfxA amino acid sequences using the UPGMA method. ResultsAmong the 45 Prevotella isolates identified, 35 (77.8%) produced β-lactamases and had the cfxA genes. The tetQ, ermF, and nim genes were detected in 53.3%, 17.8%, and 0% of the 45 isolates, respectively. Among the 33 sequenced cfxA alleles, cfxA2 (45.5%) was the most frequent, followed by cfxA3 (42.4%) and a novel variant (cfxA7, 12.1%). The novel CfxA7 β-lactamase had a novel L155F substitution not previously reported in CfxA variants. The MICs of all β-lactam agents tested, excluding cefmetazole and meropenem, were lower among cfxA7-positive isolates than in cfxA2-and cfxA3-positive isolates. ConclusionsDifferences in MICs of penicillins and cephalosporins may be due to amino acid substitutions in the CfxA variants, CfxA2, CfxA3, and CfxA7, among Prevotella isolates. Possession of cfxA-mobA, tetQ, and ermF may increase the risks of the emergence and spread of multidrug-resistant Prevotella species.

Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal.

Birds are potential carriers of pathogens affecting humans and agriculture. Aiming to evaluate the occurrence of the top three most important foodborne pathogens in free-living birds in Portugal, we investigated 108 individual fecal samples from free-living birds and one pooled sample of gull feces (n = 50) for the presence of Escherichia coli (pathogenic and non-pathogenic), Salmonella spp. and Campylobacter spp. Virulence- and antimicrobial resistance- (AMR) associated genes were detected by PCR and Whole-Genome Sequencing (WGS), and phenotypic (serotyping and AMR profiles) characterization was performed. Overall, 8.9% of samples tested positive for pathogenic E. coli, 2.8% for Salmonella spp., and 9.9% for Campylobacter spp. AMR was performed on all pathogenic isolates and in a fraction of non-pathogenic E. coli, being detected in 25.9% of them. Ten of the tested E. coli isolates were multidrug-resistant (MDR), and seven of them were Extended-spectrum β-lactamase (ESBL) producers. Among Salmonella (n = 3) and Campylobacter (n = 9), only one strain of C. jejuni was identified as MDR. Most of the identified serotypes/sequence types had already been found to be associated with human disease. These results show that free-living birds in Portugal may act as carriers of foodborne pathogens linked to human disease, some of them resistant to critically important antimicrobials.

2225. Analysis of Co-Resistance Among Escherichia coli Urine Isolates From Female Outpatients in the United States

Abstract Background In the past 10 years, there has been a substantial increase in antimicrobial resistance among uropathogens in community-acquired uncomplicated urinary tract infections (uUTIs), including extended spectrum β-lactamase producing (ESBL+) Enterobacterales and multidrug resistance. This study examined urine isolates from female outpatients in the United States (US) for co-resistance among Escherichia coli (E. coli). Methods This was a retrospective, cross-sectional study of 30-day non-duplicate E. coli urine isolates (first isolates collected within a 30-day period) from female outpatients (≥ 12 years of age) at 304 US facilities. Included patients had ≥ 3 months of data from 2011 to 2019 (Becton, Dickinson and Company [BD] Insights Research Database). Urine-isolated E. coli were defined as ESBL+ by 1) commercial panel or 2) not susceptible (NS; intermediate/resistant) to ceftriaxone, cefotaxime, ceftazidime, or cefepime), or NS to any of: fluoroquinolones (FQs), trimethoprim/sulfamethoxazole (SXT), or nitrofurantoin (NFT). Microbiological co-resistance phenotypes were characterized in isolates NS to ≥ 2 of the 4 resistance phenotypes assessed. Results In total, 856,918 unique isolates were evaluated. Co-resistance data are shown in the Table. Of ESBL+ isolates (96,306), 72.4% were co-resistant to FQ, 56.7% to SXT, and 11.9% to NFT; 6.8% had all 4 phenotypes. For FQ NS isolates (319,354), 21.8% were also ESBL+, 51.6% were co-resistant to SXT, 8.0% were co-resistant to NFT, and 2.0% had all 4 phenotypes. Among SXT NS isolates (384,304), 14.2% were also ESBL+, 42.9% were co-resistant to FQ, 6.8% were co-resistant to NFT, and 1.7% had all 4 phenotypes. Finally, for NFT NS isolates (56,954), 20.1% were ESBL+, 44.7% were co-resistant to FQ, 46.0% were co-resistant to SXT, and 11.5% had all 4 phenotypes. Table.Co-resistance phenotype combinations among urine-isolated Escherichia coli (2011–2019)In total, 856,918 non-duplicate (30-day) Escherichia coli isolates were evaluated.Note: Some isolates had overlapping susceptibilities/antimicrobial resistance types, hence individual phenotype totals do not reflect total number of isolates evaluated.Abbreviations: ESBL+, extended spectrum β-lactamase-producing (by commercial panel or not susceptible to ceftriaxone, cefotaxime, ceftazidime or cefepime); FQ, fluoroquinolone; NFT, nitrofurantoin; NS, not susceptible; SXT, trimethoprim/sulfamethoxazole. Conclusion Among urine-isolated E. coli, there was a high prevalence of co-resistance, particularly for ESBL+ isolates where co-resistance to FQ was &amp;gt; 70%. The availability of effective oral treatments for uUTI is limited by uropathogen antibiotic resistance. These data may help inform appropriate empiric prescribing practices to optimize the treatment of uUTI, mitigate multiple drug exposure for a uUTI event, and potentially influence long-term resistance patterns among E. coli. Disclosures Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Vikas Gupta, PharmD, Becton, Dickinson and Company: Employee of, and shareholder in, Becton, Dickinson and Company, and the company received funding from GlaxoSmithKline plc. to conduct this study|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 Aruni Mulgirigama, MBBS, GlaxoSmithKline plc.: Employee and shareholder|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 Ashish V. Joshi, PhD, GlaxoSmithKline plc.: Employee and shareholder|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study Nicole E. Scangarella-Oman, MS, GlaxoSmithKline plc.: Employee and shareholder Kalvin Yu, MD, FIDSA, Becton, Dickinson and Company: Employee of, and shareholder in, Becton, Dickinson and Company, and the company received funding from GlaxoSmithKline plc. to conduct this study|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 Janet Watts, PhD, Becton, Dickinson and Company: Employee of Becton, Dickinson and Company, and the company received funding from GlaxoSmithKline plc. to conduct this study|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 Fanny S. Mitrani-Gold, MPH, GlaxoSmithKline plc.: Employee and shareholder|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502.