AmpC Β-lactamase Research Articles

Carbapenem-Resistant but Cephalosporin-Susceptible Pseudomonas aeruginosa in Urinary Tract Infections: Opportunity for Colistin Sparing.

This paper briefly reports the occurrence and epidemiology of carbapenem-resistant but cephalosporin-susceptible (Car-R/Ceph-S) Pseudomonas aeruginosa isolates from urinary tract infections (UTIs) in a tertiary-care hospital in the Southern Region of Hungary, and the phenotypic characterization of the possible resistance mechanisms in these isolates. Isolates and data were collected regarding P. aeruginosa UTIs corresponding to the period between 2008 and 2017. Susceptibility testing was performed using the Kirby–Bauer disk diffusion method; minimum inhibitory concentrations (MICs) of the isolates were determined using E-tests. The phenotypic detection of ampicillin C-type (AmpC) β-lactamases, efflux pump overexpression and carbapenemase production was also performed. P. aeruginosa represented n = 575 (2.72% ± 0.64%) from outpatient, and n = 1045 (5.43% ± 0.81%) from inpatient urinary samples, respectively. Based on the disk diffusion test, n = 359 (22.16%) were carbapenem-resistant; in addition to carbapenems, n = (64.34%) were also resistant to ciprofloxacin; n = (60.17%) to gentamicin/tobramycin; n = (58.51%) to levofloxacin; and n = (27.57%) to amikacin. From among the carbapenem-resistant isolates, n = 56 (15.59%) isolates were multidrug-resistant, while n = 16 (4.46%) were extensively drug-resistant. From among the Car-R/Ceph-S isolates (n = 57), overexpression of AmpC was observed in n = 7 cases (12.28%); carbapenemase production in n = 4 (7.02%); while overexpression of efflux pumps was present in n = 31 (54.39%) isolates. To spare last-resort agents, e.g., colistin, the use of broad-spectrum cephalosporins or safe, alternative agents should be considered in these infections.

Regulation of Expression of β‐lactamase (ampC) and Lytic Transglycosylase by a Cascade of RpoE7‐>RpoH3 Sigma Factors Regulates Ampicillin Resistance in Azospirillum brasilense Sp245

Bacteria colonizing rhizosphere of plants face an intense competition for the nutrients present in the root exudates. This competition also involves production of antibiotics as well as of the enzymes that prevent inhibitory effects of antibiotics. Extra‐cytoplasmic function (ECF) sigma factors are known to initiate the transcriptions of genes involved in the adaptation against several stresses including cell envelope stress caused by antibiotics. Azospirillum brasilense is a plant growth promoting rhizobacterium, which colonizes the rhizosphere of a large number of non‐legume crops. The genome of A.brasilense Sp245 encodes 23 different sigma factors; one σ54 (RpoN), one σ70 (RpoD) and 21 alternative sigma factors of σ70 family. Out of 21 alternative sigma factors, 5 belong to RpoH, 10 to RpoE (ECF), and 6 to FecI type of σ factors. When the gene encoding RpoE7 was inactivated, the rpoE7::km mutant showed resistance to higher concentration of β‐lactam antibiotics compared to its parent. Inactivation of rpoE7 led to a decrease in the activity of its own promoter as well as of its divergently organized gene encoding a lytic‐transglycosylase. Transcription start site of rpoE7 was identified by 5’RACE, and studies with lacZ fusions with the promoters of rpoE7 and lytic transglycosylase genes indicated that both the promoters were regulated by RpoE7. Since β‐lactamases are known to be involved in conferring resistance to β‐lactams, we examined the expression of β‐lactamase paralogs in the parent and rpoE7::km mutant, and observed upregulation of one β‐lactamase (AmpC) in rpoE7::km compared to the wild type. The promoter activity of ampC and its regulator ampR were also increased in the rpoE7::km mutant. We also noted that RpoE7 regulated the expression of AmpC indirectly via RpoH3 sigma factor. Altogether, these data suggest that ampicillin resistance in A. brasilense is regulated by a regulatory cascade of RpoE7 and RpoH3.Support or Funding InformationParul Pandey, Ashutosh Prakash Dubey, Shivangi Mishra and Vijay Shankar Singh are Thankful to ICAR, CSIR, and UGC respectively for the research grant.

Clinical Evolution of AmpC-Mediated Ceftazidime-Avibactam and Cefiderocol Resistance in Enterobacter cloacae Complex Following Exposure to Cefepime.

We report 2 independent patients from whom carbapenem and ceftazidime-avibactam-resistant Enterobacter cloacae complex strains were identified. The ceftazidime-avibactam resistance was attributed to a 2-amino acid deletion in the R2 loop of AmpC β-lactamase, which concurrently caused resistance to cefepime and reduced susceptibility to cefiderocol, a novel siderophore cephalosporin.

Combined Molecular and Phenotypic Antimicrobial Susceptibility Testing Is Beneficial in Detection of ESBL and AmpC Beta-Lactamase Producing Isolates of Enterobacteriaceae in Pediatric Patients with Bloodstream Infections.

Objectives: The aim was to assess the potential advantage of combined genotypic testing with phenotypic antimicrobial susceptibility testing (AST) to detect AmpC β-lactamases (AmpC) and extended-spectrum β-lactamases (ESBL) producing Enterobacteriaceae isolated from blood cultures in a pediatric population. Materials and Methods: All first-time Enterobacteriaceae isolates recovered from blood cultures of pediatric patients at the Cincinnati Children's Hospital Medical Center between January 2017 and December 2018 were evaluated. The Check-MDR CT103XL β-lactamase assay was used to determine the presence of AmpC and ESBL, while AST was performed using the VITEK 2 platform. Phenotypic ESBL resistance was defined by resistance to either ceftriaxone or ceftazidime using Clinical and Laboratory Standards Institute breakpoints, while combined cefoxitin resistance with ceftriaxone or ceftazidime resistance was used to detect AmpCs (as per European Committee on Antimicrobial Susceptibility Testing standards). Results: Overall, there were 170 isolates. Genotypically, 21 (12.4%) had AmpC and 18 (10.6%) had ESBL genes detected. Phenotypically, 11 (6.5%) isolates were AmpC and 26 (15.3%) were ESBL producing organisms. Genotypic testing identified an additional 14 AmpC and two ESBL isolates that failed to meet phenotypic criteria. Conclusions: Using combined genotypic and phenotypic methods to detect AmpC and ESBL producing organisms increased the identification of resistant organism and provided potentially clinical relevant data to guide the treatment of resistant organisms.

Characterization of AmpC β-lactamase mutations of extensively drug-resistant Pseudomonas aeruginosa isolates that develop resistance to ceftolozane/tazobactam during therapy

IntroductionWe characterized AmpC β-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. MethodsFive pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. ResultsThe ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229–E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. ConclusionsCeftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent.

Burden of Multidrug-Resistant Acinetobacter baumannii Infection in Hospitalized Patients in a Tertiary Care Hospital of Nepal.

IntroductionAcinetobacter baumannii has emerged as a significant multidrug-resistant (MDR) nosocomial pathogen worldwide and is responsible for various healthcare-associated infections. The MDR strains have been reported increasingly during the last decades in hospitalized patients. They have developed resistance to most of the available antibiotics and are known to produce various acquired β-lactamases. The β-lactamase producing strains have a potential for rapid dissemination in hospital settings, as it is often plasmid-mediated. The Infectious Diseases Society of America (ISDA) stated A. baumannii as one of the “red alert” pathogens that greatly threatens the utility of our current antibacterial armamentarium. The study attempted to investigate the spectrum and antimicrobial resistance among MDR A. baumannii and their potential implications in hospitalized patients in a tertiary care hospital of Nepal.MethodsThis study was conducted at Tribhuvan University Teaching Hospital (TUTH), Nepal from January 2017 to December 2017. A total of 177 A. baumannii isolated from hospitalized patients were included in the study. The AST was performed by disc diffusion method. The MDR strains were identified by the criteria of Magiorakos et al, ESBL production by CLSI guidelines, and AmpC β-lactamase production by the AmpC disc test. MBL and KPC production were detected as per the method of Tsakris et al.ResultsOut of 177 A. baumannii, 91.0% were MDR isolates. Among the MDR isolates, the majority were isolated from respiratory tract specimens and were isolated from ICU patients. Most of the MDR isolates were resistant to all first-line antibiotics and all were completely sensitive to only polymyxin B and colistin sulfate. MBL (67.7%) was the common β-lactamase production among MDR isolates.ConclusionAcinetobacter baumannii can cause a vast variety of infections in hospitalized patients. The highly resistant MDR strains are common in tertiary care hospitals. This bacteria lead to high morbidity and mortality as we are left with the only option of treating them by potentially toxic antibiotics like colistin sulfate and polymyxin B. Detection of drug resistance and rational use of antibiotics play a crucial role in the fight against this MDR pathogen.

Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Isolated from Healthy Thoroughbred Racehorses in Japan.

Simple SummaryExtended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases (AmpCs) have been recognized as an emerging global problem in humans and animals. These enzymes provide a mechanism of resistance by inactivating β-lactam antibiotics and are mostly encoded on plasmids, which can be easily transmitted to other bacteria in humans, animals, and the environment. Several clinical diseases caused by Klebsiella spp. infection have been confirmed in the horse community. The emergence of antimicrobial resistance in Klebsiella spp. increases the risk of treatment failure in infected horses. In this study, we investigated the presence of ESBL/AmpC-producing Klebsiella spp. isolated from healthy Thoroughbred racehorses in Japan. The results showed that ESBL/AmpC-producing Klebsiella pneumoniae (ESBL/AmpC-KP) isolated from horses have co-resistance to other β-lactam antibiotics as multidrug-resistant (MDR) bacteria. Genetic relatedness analysis suggested that plasmid-mediated AmpC-KP clones may spread between horses. This is the first study to show K. pneumoniae carrying MDR plasmid-mediated AmpC isolated from racehorses. Continuous monitoring antimicrobial resistance to this species is required in order to control the spread of MDR ESBL/AmpC-KP in the racehorse community.Extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing Klebsiella spp. have become a major health problem, leading to treatment failure in humans and animals. This study aimed to evaluate the presence of ESBL/AmpC-producing Klebsiella spp. isolated from racehorses in Japan. Feces samples from 212 healthy Thoroughbred racehorses were collected from the Japan Racing Association Training Centers between March 2017 and August 2018. ESBL/AmpC-producing Klebsiella spp. were isolated using selective medium containing 1 µg/mL cefotaxime. All isolates were subjected to bacterial species identification (MALDI-TOF MS), antimicrobial susceptibility test (disk diffusion test), characterization of resistance genes (PCR), conjugation assay, and genetic relatedness (multilocus sequence typing/MLST). Twelve ESBL/AmpC-producing Klebsiella pneumoniae (ESBL/AmpC-KP) were isolated from 3.3% of horse samples. Antimicrobial resistance profiling for 17 antimicrobials showed all ESBL/AmpC-KP were multidrug-resistant (MDR). Only 1 isolate was confirmed as an ESBL producer (blaCTX-M-2-positive), whereas the other 11 isolates were plasmid-mediated AmpC (pAmpC) producers (blaCMY positive). On the basis of MLST analysis, the ESBL-KP isolate was identified as sequence type (ST)-133 and four different STs among AmpC-KP isolates, ST-145, ST-4830, ST-4831, and ST-4832, were found to share six of the seven loci constituting a single-locus variant. This is the first study to show K. pneumoniae carrying MDR pAmpC isolated from a racehorse.

Molecular characteristics of extended-spectrum β-lactamase/AmpC-producing Salmonella enterica serovar Virchow isolated from food-producing animals during 2010–2017 in South Korea

Global dissemination of non-typhoidal Salmonella producing extended-spectrum β-lactamase (ESBL) is a public-health concern. Recently, the prevalence of Salmonella spp. resistant to third-generation cephalosporins has been increasing in food-producing animals in Korea. In this study, we investigated resistance mechanisms and molecular characteristics of S. Virchow isolates resistant to extended-spectrum cephalosporins (ESCs). We obtained 265 S. Virchow isolates from fecal and carcasses samples of cattle (n = 2), pigs (n = 7), and chickens (n = 256) during 2010–2017, and observed high ESC-resistance (63.8%, 169/265); most of the resistant isolates (96.4%) were obtained from chickens. ESC-resistant S. Virchow isolates (n = 169) showed significantly higher resistance rates to other antimicrobials (especially aminoglycosides and tetracycline, p-value <0.0001), as well as prevalence of multidrug resistance, than did ESC-susceptible S. Virchow isolates (n = 96). All ESC-resistant S. Virchow produced CTX-M-15-type ESBL (n = 147) and/or CMY-2-type AmpC β-lactamase (n = 23). ESC-resistant S. Virchow represented seven pulsotypes, predominantly composed of type II (58.6%) and III (26.0%), detected in 69 farms in 10 provinces, and 33 farms in 7 provinces, respectively. Genes encoding ESC-resistance were horizontally transferred by conjugation to recipient E. coli J53; this was demonstrated in 28.8% (42/146) of blaCTX-M-15-positive isolates and in 50.0% (11/22) of blaCMY-2-positive isolates. All conjugative plasmids carrying blaCTX-M-15 and blaCMY-2 genes belonged to ST2-IncHI2 and ST12/CC12-IncI1, respectively. Genetic features of transferred bla genes were involved with ISEcp1 in both blaCTX-M-15 and blaCMY-2; ISEcp1 plays a critical role in the efficient capture, expression, and mobilization of bla genes. In addition to blaCTX-M-15 genes, resistance markers to aminoglycosides and/or tetracycline were co-transferred to recipient E. coli J53.Our results show a high prevalence of ESBL-producing S. Virchow in chickens and chicken carcasses. Specific blaCTX-M-15 and blaCMY-2-carrying S. Virchow clones and plasmids were predominant in food-producing animals nationwide. Restriction of antimicrobial use and proper biosecurity practices at the farm level should be urgently implemented in the poultry industry.

Evaluation of the Association Between Gastric Acid Suppression and Risk of Intestinal Colonization With Multidrug-Resistant Microorganisms

Acid suppressants inhibit gastric acid secretion and disrupt the intestinal microbiome. Whether acid suppression increases the risk of colonization with multidrug-resistant microorganisms (MDROs) is unclear. To systematically examine the association of use of acid suppressants with the risk of colonization with MDROs and to perform a meta-analysis of current evidence. PubMed, Embase, the Web of Science Core Collection, and the Cochrane Central Register of Controlled Trials were searched from database inception through July 8, 2019. Study selection was performed independently by 2 authors (R.P.J.W. and C.M.J.E.V.-G.) on the basis of predefined selection criteria; conflicts were resolved by consensus or by an adjudicator (K.v.D.). Human observational studies (case control, cohort, and cross-sectional) and clinical trial designs were selected if they quantified the risk of MDRO colonization in users of acid suppressants in comparison with nonusers. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) and Meta-analysis of Observational Studies in Epidemiology (MOOSE) recommendations were followed. Data were extracted independently by the same 2 authors, and adjudication was conducted when necessary. Risk of bias was assessed according to a modified Newcastle-Ottawa Scale. Pooled odds ratios (ORs) were estimated using random-effects models; heterogeneity was evaluated using the I2 method. The primary outcome measure was intestinal colonization with MDROs of the Enterobacterales order (producing extended-spectrum β-lactamases, carbapenemases, or plasmid-mediated AmpC β-lactamases), vancomycin-resistant enterococci, methicillin-resistant or vancomycin-resistant Staphylococcus aureus, or multidrug-resistant Pseudomonas or Acinetobacter species. A total of 26 observational studies including 29 382 patients (11 439 [38.9%] acid suppressant users) met the selection criteria. Primary meta-analysis of 12 studies including 22 305 patients that provided adjusted ORs showed that acid suppression increased the odds of intestinal carriage of MDROs of the Enterobacterales order and of vancomycin-resistant enterococci by roughly 75% (OR = 1.74; 95% CI, 1.40-2.16; I2 = 68%). The odds were concordant with the secondary pooled analysis of all 26 studies (OR = 1.70; 95% CI, 1.44-1.99; I2 = 54%). Heterogeneity was partially explained by variations in study setting and the type of acid suppression. Acid suppression is associated with increased odds of MDRO colonization. Notwithstanding the limitations of observational studies, the association is plausible and is strengthened by controlling for confounders. In view of the global increase in antimicrobial resistance, stewardship to reduce unnecessary use of acid suppressants may help to prevent MDRO colonization.

Antibiotic resistant and extended-spectrum β-lactamase producing faecal coliforms in wastewater treatment plant effluent

Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms of antibiotic resistance in the effluent of two urban WWTPs in Ireland. This information is critical to identifying the role of WWTPs in the dissemination of ARB and ARGs into the environment. Effluent samples were collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried out to identify AmpC or extended-spectrum β-lactamase (ESBL) producers. The presence of ESBL genes were detected by PCR. Plasmids carrying ESBL genes were transformed into Escherichia coli DH5α recipient and underwent plasmid replicon typing to identify incompatibility groups. More than 90% of isolated faecal coliforms were resistant to amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin (up to 31.62%) and imipenem (up to 15.93%) were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms expressed a multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 5.22% of isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates carried blaTEM, 1.4% blaSHV-12, 0.2% blaCTX-M-1 and 1% blaCTX-M-15). Plasmids extracted from 52 ESBL isolates were successfully transformed into recipient E. coli. The detected plasmid incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms and are sources of ESBL-producing, carbapenem and colistin resistant Enterobacteriaceae.

A Standard Numbering Scheme for Class C β-Lactamases.

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.

Integrated Genome-Wide Analysis of an Isogenic Pair of Pseudomonas aeruginosa Clinical Isolates with Differential Antimicrobial Resistance to Ceftolozane/Tazobactam, Ceftazidime/Avibactam, and Piperacillin/Tazobactam.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the main causes of morbidity and mortality in hospitalized patients and the leading cause of nosocomial infections. We investigated, here, two MDR P. aeruginosa clinical isolates from a hospitalized patient with differential antimicrobial resistance to ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), and piperacillin/tazobactam (P/T). Their assembled complete genomes revealed they belonged to ST235, a widespread MDR clone; and were isogenic with only a single nucleotide variant, causing G183D mutation in AmpC β-lactamase, responsible for a phenotypic change from susceptible to resistant to CZA and C/T. Further epigenomic profiling uncovered two conserved DNA methylation motifs targeted by two distinct putative methyltransferase-containing restriction-modification systems, respectively; more intriguingly, there was a significant difference between the paired isolates in the pattern of genomic DNA methylation and modifications. Moreover, genome-wide gene expression profiling demonstrated the inheritable genomic methylation and modification induced 14 genes being differentially regulated, of which only toxR (downregulated), a regulatory transcription factor, had its promoter region differentially methylate and modified. Since highly expressed opdQ encodes an OprD porin family protein, therefore, we proposed an epigenetic regulation of opdQ expression pertinent to the phenotypic change of P. aeruginosa from resistant to susceptible to P/T. The disclosed epigenetic mechanism controlling phenotypic antimicrobial resistance deserves further experimental investigation.

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation.

To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC β-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum β-lactamase possessing weak carbapenemase activity, encoded by another plasmid. We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

Extended-spectrum beta-lactamases and plasmid diversity in urinary isolates of Escherichia coli in Croatia: a nation-wide, multicentric, retrospective study.

In recent years, a dramatic increase in the prevalence of Escherichia coli strains producing extended-spectrum β-lactamases (ESBLs) has been observed-both in the community and in healthcare settings. This multicentric study aimed to characterize ESBLs produced by E. coli isolates causing hospital-onset and community urinary tract infections, as well as to compare their antimicrobial sensitivity patterns, β-lactamase content and plasmid types. Phenotypic tests for the detection of ESBLs and plasmid-mediated AmpC β-lactamases were initially pursued, followed by molecular detection of resistance genes, plasmid characterization, genotyping with pulsed-field gel electrophoresis and whole genome sequencing (WGS). The isolates exhibited high level of resistance to expanded-spectrum cephalosporins (ESC) and carried CTX-M (cefotaximase-Munich) or TEM (Temoniera) β-lactamases. All six representative isolates subjected to WGS belonged to the widespread clone ST131. In conclusion, our study demonstrated dissemination of group 1 CTX-M positive E. coli in different geographic regions of Croatia, but also different components of the health care systems (hospitals, nursing homes and the community) and confirmed the switch from SHV-2 (suphydril variant) and SHV-5 ESBLs to the nation-wide predominance of group 1 CTX-M β-lactamases. Different plasmids were shown to be associated with the dissemination of blaCTX-M genes in different geographic regions of Croatia.

Prevalence and Molecular Characteristics of Extended-Spectrum and AmpC β-Lactamase Producing Escherichia coli in Grazing Beef Cattle.

The emergence of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase producing Escherichia coli represent a contemporary public health threat. ESBL and AmpC β-lactamase genes translocate between chromosomes and plasmids, facilitating rapid spread throughout the environment. In this study, ESBL/AmpC producing bacteria were isolated from beef cattle farms with seldom antibiotic use. Eleven farms out of 17 tested, had ESBL/AmpC producing E. coli in animals, soil, and forage samples. Fifty-nine CTX-M or CMY-2 positive E. coli isolates were further characterized with whole-genome sequencing. The isolates commonly carried CMY-2, TEM, or CTX-M genes, and over half encoded both CTX-M and TEM genes. Using comparative genomics, antimicrobial resistance genes from 12 classes of antimicrobial were identified and confirmed by antibiotic susceptibility test, revealing multidrug resistance against multiple classes of antibiotics. Virulence factors related to adherence, invasion, iron uptake, and bacterial secretion systems were shared by all isolates; some of which were identified as enteropathogenic E. coli. Phylogenetic analyses revealed a pattern of close genetic relatedness, suggesting that ESBL/AmpC producing E. coli were transmitted among farms as well as independent evolution within farms. Our results indicate that ESBL and AmpC β-lactamases prevail in food animal production system regardless antibiotic use and have the characteristics for zoonotic transmission.

Piperacillin-Tazobactam-Resistant/Third-Generation Cephalosporin-Susceptible Escherichia coli and Klebsiella pneumoniae Isolates: Resistance Mechanisms and In vitro-In vivo Discordance

We previously reported the detection of Escherichia coli and Klebsiella pneumoniae that displayed in vitro piperacillin-tazobactam (TZP) resistance but were susceptible to third-generation cephalosporins (TZP-R/Ceph3-S). In this study, we assessed the phenotypic and genotypic profiles of 12 clinical non-clonal TZP-R/Ceph3-S E. coli and K. pneumoniae isolates derived from bloodstream infections. Whole-genome sequencing revealed that most of the TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined harbored blaTEM-1 and blaSHV-1 genes, respectively, but none harbored extended-spectrum β-lactamase, AmpC β-lactamase or carbapenemase genes. Increasing the tazobactam concentration from 4 mg/L to 16 mg/L restored TZP in vitro susceptibility among E. coli isolates expressing TEM-1, but had minimal impact on the susceptibility of K. pneumoniae to TZP. Real-time qPCR analysis showed that blaTEM-1 expression was amplified in TZP-R E. coli upon incubation with sub-inhibitory TZP concentrations. Using an immunocompetent murine septicemia model, the efficacy of TZP against TZP-R/Ceph3-S isolates was assessed using TZP doses that mimicked human plasma exposures following intravenous (IV) administration of TZP 4.5 g q6h over 0.5 h for 24h. Efficacy was assessed by survival through 96h. There was high mortality in untreated control mice for all tested isolates. Compared with controls, TZP human-simulated exposure significantly improved survival for all TZP-R/Ceph3-S E. coli and K. pneumoniae isolates examined (P<0.05). Thus, TZP was associated with remarkable in vivo activity against TZP-R/Ceph3-S E. coli and K. pneumoniae despite the observed resistance in vitro.

Beta-lactamase Profile and Biofilm Production of Pseudomonas aeruginosa Isolated from a Tertiary Care Hospital in Kolkata, India

Introduction: Nosocomial infections caused by Multidrug Resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) have become a major clinical and public health concern. Moreover, the biofilm production protects the bacteria from antibiotics and thereby makes the drugs ineffective. Aim: To find out the β-lactamases profile of antimicrobial resistance and biofilm production of P. aeruginosa isolated from different clinical specimens of patients attending a tertiary care hospital of Kolkata, West Bengal, India. Materials and Methods: A total of 394 consecutive, non-duplicate isolates of P. aeruginosa were identified from 3559 Gram negative bacilli over a period of two years from July 2016 to June 2018. Identification of the isolates and antibiotic sensitivity testing was performed by using automated method and interpreted. Extended Spectrum β-Lactamases (ESBLs), Amp-C β-lactamase (AmpC) and Metallo-β-Lactamases (MBLs) were phenotypically detected by disk synergy test and MBL-encoding genes were detected by multiplex Polymerase Chain Reaction (PCR). Biofilm production was done by tissue culture plate assay. Laboratory data and test results were statistically analysed in counts and percentages using MS Excel 2010 version. Results: Out of 394 strains of P. aeruginosa 288 (73.10%) were isolated from male patients whereas 106 (26.90%) were isolated from female patients. Maximum number of cases (67.26%) were from adult populations. The resistance pattern showed 72.33% resistance to ticarcillin-clavulanic acid followed by ciprofloxacin (53.80%), levofloxacin (50.25%), gentamicin (51.52%), ceftazidime (CAZ) (45.93%), cefoperazone-sulbactam (40.1%), aztreonam (34.77%), imipenem (33.5%), piperacillin/tazobactum (30.96%) and Meropenem (MRP) (29.18%). Out of 394 Pseudomonas spp. isolates, 204 (51.77%) were MDR. Overall, ESBLs, AmpC and carbapenemase (MBL) production was detected in 145 (36.80%), 51 (12.94%) and 49 (12.43%) isolates by phenotypic methods. By genotypic method 53.34% were carbapenemase producing and blaNDM-1 (68.75%) was the most prevalent carbapenemase gene detected followed by blaVIM (18.75%) and co-production of blaNDM-1+ blaVIM was 12.5%. Biofilm production was observed in 158 (40.10%) isolates. Conclusion: Early detection of these β-lactamases production is crucial not only for epidemiological study and effective infection control practices to limit the spread of infection but also for planning appropriate therapy according to the resistance mechanisms of the MDR strains.

Investigation of the in vitro antimicrobial activity of triclosan-coated suture material on bacteria commonly isolated from wounds in dogs.

To investigate in vitro effects of triclosan coating of suture materials on the growth of clinically relevant bacteria isolated from wounds in dogs. 6 types of suture material and 10 isolates each of methicillin-susceptible Staphylococcus pseudintermedius, methicillin-resistant S pseudintermedius, Escherichia coli, and AmpC β-lactamase and extended-spectrum β-lactamase-producing E coli from clinical wound infections. Isolates were cultured on Mueller-Hinton agar with 3 types of triclosan-coated suture, uncoated counterparts of the same suture types, and positive and negative controls. Zones of inhibition (ZOIs) were measured after overnight incubation. Sustained antimicrobial activity assays were performed with susceptible isolates. The ZOI measurements and durations of sustained antimicrobial activity were compared among suture types and isolates by statistical methods. Suture surface characteristics and bacterial adherence were evaluated qualitatively with scanning electron microscopy. ZOIs were generated only by triclosan-coated materials; triclosan-coated suture had sustained antimicrobial activity (inhibition) for 3 to 29 days against all tested pathogens. The ZOIs around triclosan-coated suture were significantly greater for S pseudintermedius isolates than for E coli isolates. Bacterial adherence to uncoated polyglactin-910 was greatest, followed by triclosan-coated polyglactin-910, and then uncoated monofilament sutures, with least adherence to coated monofilament sutures. Surface characteristics of suture materials may be as important or more important than triclosan coating for microbial inhibition; however, triclosan coating appeared to affect bacterial adherence for multifilament sutures. Triclosan-coated, particularly monofilament, sutures inhibited pathogens commonly isolated from wounds of dogs, including multidrug-resistant bacteria. Further studies are required to assess clinical efficacy of triclosan-coated suture materials in vivo.

Antibiotic resistance and extended-spectrum β-lactamase in Escherichia coli isolates from imported 1-day-old chicks, ducklings, and turkey poults.

Aim:Antimicrobial resistance is a global health threat. This study investigated the prevalence of Escherichia coli in imported 1-day-old chicks, ducklings, and turkey poults.Materials and Methods:The liver, heart, lungs, and yolk sacs of 148 imported batches of 1-day-old flocks (chicks, 45; ducklings, 63; and turkey poults, 40) were bacteriologically examined for the presence of E. coli.Results:We isolated 38 E. coli strains from 13.5%, 6.7%, and 5.4% of imported batches of 1-day-old chicks, ducklings, and turkey poults, respectively. They were serotyped as O91, O125, O145, O78, O44, O36, O169, O124, O15, O26, and untyped in the imported chicks; O91, O119, O145, O15, O169, and untyped in the imported ducklings; and O78, O28, O29, O168, O125, O158, and O115 in the imported turkey poults. The E. coli isolates were investigated for antibiotic resistance against 16 antibiotics using the disk diffusion method and were found resistant to cefotaxime (60.5%), nalidixic acid (44.7%), tetracycline (44.7%), and trimethoprim-sulfamethoxazole (42.1%). The distribution of extended-spectrum β-lactamase (ESBL) and ampC β-lactamase genes was blaTEM (52.6%), blaSHV (28.9%), blaCTX-M (39.5%), blaOXA-1 (13.1%), and ampC (28.9%).Conclusion:Imported 1-day-old poultry flocks may be a potential source for the dissemination of antibiotic-resistant E. coli and the ESBL genes in poultry production.

Antibiotic Resistance Pattern and Frequency of PER-1, SHV-1 and AMPC Type B-Lactamase Genes in Pseudomonas aeruginosa Isolated from Clinical Samples

Background: The existence of Extended Spectrum B-lactamase (ESBL) genes plays an important role in spreading B-lactam antibiotic resistance in the producing strains of these enzymes. The resistance of gram-negative bacteria, such as Pseudomonas aeruginosa, to different antimicrobial agents, especially B-lactams, has increasingly been reported. Objective: This study was conducted to determine the prevalence of TEM-1and VEB-1 beta-lactamases gene in P. aeruginosa isolates through Polymerase Chain Reaction (PCR) method. Methods: 100 clinical isolates of P. aeruginosa were collected from different clinical samples. The antibiotic susceptibility was examined by the disc diffusion method. The presence of PER-1, SHV-1 and AMPC genes was detected by PCR method. Results: Out of the studied P. aeruginosa isolates, 7, 9 and 37 isolates were positive for PER-1, SHV-1 and AMPC B-lactamases resistance genes, respectively. Patients with urinary infection had the most resistant isolates. All isolates (100%) were sensitive to polymyxin B. Conclusion: Antibiotic resistance in isolates of Pseudomonas can be caused by B-lactamases resistance genes. Noticing the increasing rate of the ESBLs producing strains, using the appropriate treatment protocol based on the antibiogram pattern of the strains is highly recommended.