Extended-spectrum Beta-lactamase Genes Research Articles

Diversity of Extended Spectrum Beta-lactamase (ESBL) genes in Escherichia coli isolated from wastewater generated by a Sick Bay located in a University Health Care Facility

BackgroundAntibiotic resistance is an ill which has ravaged so many countries of the world, the worst hit being African countries. This study investigated the occurrence of extended spectrum beta-lactamase producing E. coli and their genes in wastewater generated by the sick bay of a University Health Centre Facility. MethodsWastewater from three channels in the sick bay were collected over a period of four months for the isolation of E. coli on CHROMAgar E. coli amended with 6 μg/mL of cefotaxime. Molecular characterization of the isolates was done by detecting the presence of uidA gene. Screening for ESBL production was done using Double Disk Synergy Method, and antibiotic susceptibility testing was done using the disc diffusion method. Detection of ESBL genes was done using quantitative PCR. ResultsTwenty-four ESBL positive E. coli possessing the uidA gene were obtained from a pool of thirty-eight E. coli obtained. There was 100% resistance to sulfonamide, ertapenem, cefpodoxime and cefotaxime by the isolates, while the resistance to tetracycline was 87.5%, ciprofloxacin (20.8%), ceftazidime (75.0%) and amoxicillin-clavulanate (58.3%). The twenty-four isolates possessed blaCTX-M-1 and blaCTX-M-9, with fourteen possessing blaCTX-M-2,blaCTX-M-8 and blaCTX-M-25. Nineteen isolates harboured blaTEM, while blaCMY-1 was detected in five isolates. One isolate possessed ampC gene. blaKPC,blaNDM-1 and blaSHV were not detected in any isolate. ConclusionThere is a need for adequate treatment of wastewater from hospital/clinical settings to prevent a public health breakdown as a result of the discharge of untreated wastewater laden with antibiotic resistant bacteria and their genes into the environment.

Extended Spectrum Beta-Lactamases (ESBLs)-producing Escherichia coli and Klebsiella pneumoniae among asymptomatic Out-patients in a University Health Centre

Background: Asymptomatic carriage and spread of Extended-Spectrum Beta-Lactamase (ESBLs)-producing Enterobacteriaceae in the community are potential risk factors for transmission of infection. Objective: To determine the prevalence of ESBL resistant genes in Escherichia coli and Klebsiella pneumoniae isolated from asymptomatic out-patients. Methods: Using a questionnaire, demographic information, medical history, previous hospitalization and antibiotics used were obtained. Stool and urine samples were collected from 350 participants, cultured, and the susceptibility of the isolates to antibiotics and ESBL production were determined using the disk diffusion method. ESBL genes such as blaTEM, blaCTX, and blaSHV were identified using the Polymerase Chain Reaction. Results: Escherichia coli and Klebsiella pnuemoniae were identified from the stool samples (256; 69.9% and 89; 24.4% respectively) and urine samples (15; 4.1% and 6; 1.6% respectively). The isolates were susceptible to imipenem (330; 90.6%) and nitrofurantoin (307; 80.4%), most of the isolates were resistant to fluoroquinolones, cephalosporins, and aminoglycosides while all the isolates were resistant to ampicillin. The prevalence of ESBL was 29 (8.3%) and was observed in Escherichia coli (19; 7.0%) and Klebsiella pneumoniae (11; 12.0%), including a dual carriage. The ESBL carriers were resistant to the cephalosporins, fluoroquinolones and aminoglycosides. CTX-M (20; 66.7%), TEM (14; 46.7%), CTX-M and TEM genes co-existed in 9 (30.0%) while no SHV gene was detected in the isolates. Age, sex, prior hospitalization and antibiotics use did not predispose to ESBL carriage. Conclusion: Asymptomatic carriage of ESBL producing enterobacteria in the participants indicates that they can serve as a reservoir of the gene encoding for antibiotic resistance.

Antimicrobial Resistance Traits of Escherichia coli Isolated from Dairy Manure and Freshwater Ecosystems Are Similar to One Another but Differ from Associated Clinical Isolates.

Antimicrobial resistance (AMR) is a prevalent global health problem across human and veterinary medicine. The One Health approach to AMR is necessary to mitigate transmission between sources of resistance and decrease the spread of resistant bacteria among humans, animals, and the environment. Our primary goal was to identify associations in resistance traits between Escherichia coli isolated from clinical (n = 103), dairy manure (n = 65), and freshwater ecosystem (n = 64) environments within the same geographic location and timeframe. Clinical E. coli isolates showed the most phenotypic resistance (47.5%), followed by environmental isolates (15.6%) and manure isolates (7.7%), with the most common resistances to ampicillin, ampicillin-sulbactam, and cefotaxime antibiotics. An isolate subset was screened for extended spectrum beta-lactamase (ESBL) production resulting in the identification of 35 ESBL producers. The most common ESBL gene identified was blaTEM-1. Additionally, we found nine different plasmid replicon types including IncFIA-FIB, which were frequently associated with ESBL producer isolates. Molecular phylotyping revealed a significant portion of clinical E. coli were associated with phylotype B2, whereas manure and environmental isolates were more diverse. Manure and environmental isolates were significantly different from clinical isolates based on analyzed traits, suggesting more transmission occurs between these two sources in the sampled environment.

A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL, and AmpC genes

Resistance to third-generation cephalosporins and carbapenems in Gram-negative bacteria is chiefly mediated by beta-lactamases including extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase enzymes. Routine phenotypic detection methods do not provide timely results, and there is a lack of comprehensive molecular panels covering all important markers. An ESBL/carbapenemase high-resolution melt analysis (HRM) assay (SHV, TEM, CTX-M ESBL families, and NDM, IMP, KPC, VIM and OXA-48–like carbapenemases) and an AmpC HRM assay (16S rDNA control, FOX, MOX, ACC, EBC, CIT, and DHA) were designed and evaluated on 111 Gram-negative isolates with mixed resistance patterns. The sensitivity for carbapenemase, ESBL, and AmpC genes was 96.7% (95% confidence interval [CI]: 82.8–99.9%), 93.6% (95% CI: 85.7–97.9%), and 93.8% (95% CI: 82.8–98.7%), respectively, with a specificity of 100% (95% CI: 95.6–100%), 93.9% (95% CI: 79.8–99.3%), and 93.7% (95% CI: 84.5–98.2%). The HRM assays enable the simultaneous detection of the 14 most important ESBL, carbapenemase, and AmpC genes and could be used as a molecular surveillance tool or to hasten detection of antimicrobial resistance for treatment management.

Surveillance of OXA-244-producing Escherichia coli and epidemiologic investigation of cases, Denmark, January 2016 to August 2019.

BackgroundCarbapenemase-producing Escherichia coli are increasing worldwide. In recent years, an increase in OXA-244-producing E. coli isolates has been seen in the national surveillance of carbapenemase-producing organisms in Denmark.AimMolecular characterisation and epidemiological investigation of OXA-244-producing E. coli isolates from January 2016 to August 2019.MethodsFor the epidemiological investigation, data from the Danish National Patient Registry and the Danish register of civil registration were used together with data from phone interviews with patients. Isolates were characterised by analysing whole genome sequences for resistance genes, MLST and core genome MLST (cgMLST).ResultsIn total, 24 OXA-244-producing E. coli isolates were obtained from 23 patients. Among the 23 patients, 13 reported travelling before detection of the E. coli isolates, with seven having visited countries in Northern Africa. Fifteen isolates also carried an extended-spectrum beta-lactamase gene and one had a plasmid-encoded AmpC gene. The most common detected sequence type (ST) was ST38, followed by ST69, ST167, ST10, ST361 and ST3268. Three clonal clusters were detected by cgMLST, but none of these clusters seemed to reflect nosocomial transmission in Denmark.ConclusionImport of OXA-244 E. coli isolates from travelling abroad seems likely for the majority of cases. Community sources were also possible, as many of the patients had no history of hospitalisation and many of the E. coli isolates belonged to STs that are present in the community. It was not possible to point at a single country or a community source as risk factor for acquiring OXA-244-producing E. coli.

MOLECULAR EPIDEMIOLOGY AND HOSPITAL SPREAD OF CEFTRIAXONE NON‐SUSCEPTIBLE ENTEROBACTERALES IN THE SUNSHINE COAST REGION, AUSTRALIA

Background: Extended spectrum beta‐lactamase (ESBL) and AmpC‐producing Enterobacterales are prevalent worldwide. Poor clinical outcomes, including death, are more common in patients both colonised and infected with these organisms1. Australia is thought to have a comparatively low prevalence of such organisms, however no clear continuous surveillance system exists2. Specific ESBL type has shown significant geographic variation, having different therapeutic and infection control implications for different regions3. Little is known about the prevalence, molecular type and spread of ESBL and AmpC‐producing Enterobacterales among hospitalised patients in the Sunshine Coast region. Aims: To (i) Determine the prevalence and molecular epidemiology of ESBL and AmpC‐producing Enterobacterales, (ii) Develop an in‐house multiplex polymerase chain reaction (PCR) assay to detect common ESBL genes, (iii) Compare our PCR assay to the double disc diffusion method for detection of ESBLs, and to (iv) Establish, with whole genome sequencing (WGS), whether transmission was occurring in our intensive care unit (ICU). Methods: Clinical specimens from adult and paediatric patients culturing Enterobacterales resistant to a third‐generation cephalosporin were collected prospectively from July 2017 to August 2018 at the Sunshine Coast University Hospital laboratory. Antibiotic susceptibility testing was determined using VITEK‐2®. A validated in‐house pentaplex PCR assay was developed and used to detect common beta‐lactamase genes (CTX‐1, CTX‐9, SHV, TEM, CMY). All isolates collected from patients admitted to the ICU were identified and underwent WGS analysis using the Illumina® platform. Results: 295 clinical specimens were identified. Escherichia coli (78.8%), Klebsiella pneumoniae (16.8%) with 1‐2% consisting of Enterobacter spp., Serratia marcescens and Citrobacter freundii. Specimens included urine (53.8%), rectal swab (39.4%), pus (3.0%) and blood (2.4%). Carbapenem resistance was observed in 3.3%. ICU patient specimens made up 18.2%. The majority (87.8%) of isolates had at least one gene identified on PCR testing of which 48.5% produced CTX‐1, 31.2% produced CTX‐9, 15.9% produced SHV, 12.5% produced CMY and 40.6% produced TEM. Approximately forty percent of isolates produced more than one ESBL with the most common being CTX‐1 and TEM coproduction. A small amount (<5%) of isolates were negative on PCR testing. Fourteen (4.7%) isolates that were negative on double disc diffusion had ESBL genes detected by our PCR assay. WGS of non‐identical ICU patient E. coli (n = 44) and K. pneumoniae (n = 10) isolates revealed no strong evidence for transmission of these organisms between patients during this time period, with the smallest single nucleotide polymorphism (SNP) difference being 300 SNPs. Conclusion: Multidrug resistant Gram‐negative organisms are present on the Sunshine Coast and cause significant morbidity. Our real‐time pentaplex PCR assay was able to successfully identify common beta‐lactamase genes, revealing a frequency distribution among our organisms similar to that noted in previous Australian studies (CTX‐1 group most commonly identified). We were not able to identify evidence of ICU transmission, which recognises the success of current infection control practices at our institution.

Molecular epidemiology of Salmonella Infantis in Europe: insights into the success of the bacterial host and its parasitic pESI-like megaplasmid

Salmonella Infantis is one of the five serovars most frequently causing human salmonellosis in Europe, mainly associated with poultry. A clone harbouring a conjugative plasmid of emerging S. Infantis (pESI)-like megaplasmid, carrying multidrug resistant (MDR) and extended-spectrum beta-lactamases (ESBL) genes, has spread in the Italian broiler chicken industry also causing human illness. This work is aimed at elucidating the molecular epidemiology of S. Infantis and pESI-like in Europe using whole-genome sequencing and bioinformatics analysis, and to investigate the genetic relatedness of S. Infantis clones and pESI-like from animals, meat, feed and humans provided by institutions of nine European countries. Two genotyping approaches were used: chromosome or plasmid SNP-based analysis and the minimum spanning tree (MST) algorithm based on core-genome multilocus sequence typing (cgMLST). The European S. Infantis population appeared heterogeneous, with different genetic clusters defined at core-genome level. However, pESI-like variants present in 64.1 % of the isolates were more genetically homogeneous and capable of infecting different clonal lineages in most of the countries. Two different pESI-like with ESBL genes (n=82) were observed: bla CTX-M-1-positive in European isolates and bla CTX-M-65-positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals (merA) and disinfectants (qacEΔ). Worryingly, 66 % of the total isolates studied presented different gyrA chromosomal point mutations associated with (fluoro)quinolone resistance (MIC range 0.125–0.5 mg/L), while 18 % displayed transferable macrolide resistance mediated by mph, mef and erm(B) genes. Proper intervention strategies are needed to prevent further dissemination/transmission of MDR S. Infantis and pESI-like along the food chain in Europe.

Virulence characterization of Klebsiella pneumoniae and its relation with ESBL and AmpC beta-lactamase associated resistance

Trend analysis reveals that Klebsiella pneumoniae has witnessed a steep enhancement in the antibiotic resistance and virulence over the last few decades. The present investigation aimed at a comprehensive approach investigating antibiotic susceptibility including, extended spectrum beta-lactamase (ESBL) and AmpC β-lactamase (AmpC) resistance and the prevalence of virulence genes among the K. pneumoniae isolates. Sixty-one K. pneumoniae isolates were obtained from various clinical infections. Antimicrobial susceptibility was performed by disk diffusion method. The Mast® D68C test detected the presence of ESBLs and AmpCs phenotypically, and later presence of ESBL and AmpC genes was observed by polymerase chain reaction (PCR). Multiplex-PCR was performed to investigate various virulence genes. Amongst 61 K. pneumoniae isolates, 59% were observed as ESBL and 14.7% as AmpC producers. All ESBL producers were positive for bla CTX-M-15 , while bla CTX-M-14 was observed in 54.1% isolates. The frequency of AmpC genes was as follows: bla CMY-2 (60.7%) and bla DHA-1 (34.4%). The most frequent virulence genes were those encoding enterobactin and lipopolysaccharide. Presence of mrkD was associated with bla DHA-1 gene, while bla CMY-2 significantly (p≤0.05) correlated with the presence of iutA and rmpA virulence genes. bla DHA-1 positive isolates had urine as a significant source, while bla CMY-2 positive isolates were mainly collected from wound exudates (p≤0.05). Our results highlight that ESBL and AmpC production along with a plethora of virulence trait on K. pneumoniae should be adequately considered to assess its pathogenesis and antibiotic resistance.

Diversity, virulence and antibiogram traits of Escherichia coli recovered from potable water sources in Gharbia, Egypt.

This study aimed to assess the public health risk of coliforms and Escherichia coli contamination of potable water sources in Egypt. A total of 150 water samples (100 tap and 50 well) were collected from five districts in Gharbia governorate, Egypt. High rates of coliforms contamination were recorded in 52 and 76% of examined tap and well water samples, respectively. E. coli strains were detected in 16% of the water samples (15% tap water and 18% well water; 23.7% rural and 8.1% urban). Rural water sources were 3.5 times more likely to be contaminated than urban sources (P = 0.01). Eight (33.3%) E. coli isolates were Shiga toxin-producing E. coli (STEC). Multiple drug resistance (MDR) was observed for 62.5% of the isolates. Seven (29.2%) E. coli isolates harboured at least one of the extended-spectrum beta-lactamase (ESBL) genes. The majority (87.5%) of the STEC isolates were MDRs and harboured ESBL genes. STEC isolates were significantly more likely to resist six classes of antibiotics than non-STEC isolates. This is the first report of potable water contamination with MDR-STEC in Egypt. This study highlights an alarming public health threat that necessitates preventive interventions for public and environmental safety.

Spread of multidrug-resistant IncHI1 plasmids carrying ESBL gene blaCTX-M-1 and metabolism operon of prebiotic oligosaccharides in commensal Escherichia coli from healthy horses, France

The objective of the study was to identify the genetic determinants and characteristics of expanded-spectrum cephalosporin (ESC) resistance in commensal Escherichia coli from healthy horses in France in 2015. Faecal samples from 744 adult horses were screened for ESC-resistant E. coli isolates. The extended-spectrum beta-lactamase (ESBL)/AmpC resistance genes were identified using polymerase chain reaction (PCR) and sequencing. ESC phenotypes were horizontally transferred by conjugation or transformation. Plasmids carrying ESBL/AmpC genes were typed by PCR-based replicon typing, restriction fragment length polymorphism (RFLP), and plasmid multilocus sequence typing (pMLST). The ESC-resistant E. coli isolates were typed by XbaI macrorestriction analysis. Sixteen of 41 stables harboured at least one horse carrying ESC-resistant E. coli. The proportion of individually tested horses carrying ESC-resistant E. coli was 8.5% (28/328). Fifty non-redundant ESC-resistant E. coli isolates showing a great diversity of XbaI macrorestriction profiles belonged mainly to phylogroup B1, and were negative for major E. coli virulence genes, indicating they are commensal isolates. ESBL blaCTX-M genes were dominant (blaCTX-M-1, n=34; blaCTX-M-2, n=8; blaCTX-M-14, n=2) and located on conjugative plasmids belonging to various incompatibility groups (IncHI1, IncI1, IncN, IncY, or non-typeable). Among these, the multidrug-resistant IncHI1-pST9 plasmids were dominant and simultaneously harboured the blaCTX-M-1/2 genes and an operon enabling the metabolism of short-chain fructo-oligosaccharides (scFOS). In conclusion, commensal E. coli of French horses displayed a significant distribution of IncHI1-pST9 plasmids carrying both the blaCTX-M-1/2 gene and the fos metabolism operon. This finding highlights the risk of co-selection of multidrug-resistant IncHI1 plasmids carrying ESBL genes possibly mediated by the use of scFOS as prebiotic in horses.

A survey of extended-spectrum beta-lactamase-producing Enterobacteriaceae in urban wetlands in southwestern Nigeria as a step towards generating prevalence maps of antimicrobial resistance.

In many countries, emission of insufficiently treated wastewater into water bodies appears to be an important factor in spreading clinically relevant antimicrobial resistant bacteria. In this study, we looked for the presence of Enterobacteriaceae strains with resistance to 3rd generation cephalosporin antibiotics in four urban wetlands in southwestern Nigeria by isolation, whole genome sequencing and qPCR enumeration of marker genes. Genome analysis of multi-drug resistant and potentially pathogenic Escherichia coli isolates (members of the widely distributed ST10 complex) revealed the presence of the extended spectrum beta-lactamase gene blaCTX-M-15 on self-transmissible IncF plasmids. The gene was also present together with a blaTEM-1B gene on self-transmissible IncH plasmids in multi-drug resistant Enterobacter cloacae isolates. A Citrobacter freundii isolate carried blaTEM-1B on an IncR-type plasmid without discernable conjugation apparatus. All strains were isolated from a wetland for which previous qPCR enumeration of marker genes, in particular the ratio of intI1 to 16S rRNA gene copy numbers, had indicated a strong anthropogenic impact. Consistent with the isolation origin, qPCR analysis in this study showed that the blaCTX-M gene was present at an abundance of 1x10-4 relative to bacterial 16S rRNA gene copy numbers. The results indicate that contamination of these urban aquatic ecosystems with clinically relevant antibiotic resistant bacteria is substantial in some areas. Measures should therefore be put in place to mitigate the propagation of clinically relevant antimicrobial resistance within the Nigerian aquatic ecosystems.

Epidemic spread of IncI1/pST113 plasmid carrying the Extended-Spectrum Beta-Lactamase (ESBL) blaCTX-M-8 gene in Escherichia coli of Brazilian cattle

IntroductionThe prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide and the Agri-Food sector acts as a reservoir of clinically relevant ESBL genes. Our study aimed at detecting and characterizing ESBL-producing Enterobacteriaceae responsible for intestinal colonization of Brazilian bovines. Material and methodsESBL-producing E. coli isolates were recovered from fecal samples of healthy cattle in Northwest Brazil. Antimicrobial susceptibility was determined by disk diffusion. Resistance and virulence genes were identified by PCR and amplicons were sequenced, clonality was assessed by PFGE and MLST, and plasmids were characterized by replicon typing, S1-PFGE and Southern blot hybridizations. Transferability of ESBL genes was assessed by conjugation assay. ResultsA total of 40 ESBL-producing E. coli were characterized, which originated from 34/191 animals (17.8 %) and 15/22 farms (68.2 %). The blaCTX-M-8 gene was the most frequent ESBL gene (62.5 %), followed by blaSHV-2a (20.0 %), blaCTX-M-2 (10.0 %), and blaCTX-M-15 (7.5 %). The blaCTX-M-8 gene was localized on the IncI1/pST113 plasmid in multiple E. coli sequence types across unrelated animals and farms. DiscussionWe report the first characterization and a high prevalence of ESBL-producing E. coli in the beef cattle sector in Brazil, which is mainly supported by the spread of an epidemic IncI1/pST113/blaCTX-M-8 plasmid. Since Brazil is one of the biggest beef meat exporters worldwide, the spread of this ESBL plasmid across other sectors, countries and continents should be considered with attention.

The Impact of Direct-Fed Microbials and Phytogenic Feed Additives on Prevalence and Transfer of Extended-Spectrum Beta-Lactamase Genes in Broiler Chicken

Poultry frequently account for the highest prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in livestock. To investigate the impact of direct-fed microbials (DFM) and phytobiotic feed additives on prevalence and conjugation of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, an animal trial was conducted. Lactobacillus agilis LA73 and Lactobacillus salivarius LS1 and two commercial phytogenic feed additives (consisting of carvacrol, cinnamaldehyde, and eugenol) were used as feed additives either alone or as a combination of DFM and phytogenic feed additive. An ESBL-producing E. coli donor and a potentially pathogenic Salmonella Typhimurium recipient were inoculated at 5 × 109 cells/mL in cecal contents from 2-week-old broilers. Conjugation frequencies were determined after 4 h aerobic co-incubation at 37 °C and corrected for the impact of the sample matrix on bacterial growth of donor and recipient. Surprisingly, indigenous Enterobacteriaceae acted as recipients instead of the anticipated Salmonella recipient. The observed increase in conjugation frequency was most obvious in the groups fed the combinations of DFM and phytogenic product, but merely up to 0.6 log units. Further, cecal samples were examined for ESBL-producing Enterobacteriaceae on five consecutive days in broilers aged 27–31 days. All samples derived from animals fed the experimental diet showed lower ESBL-prevalence than the control. It is concluded that Lactobacillus spp. and essential oils may help to reduce the prevalence of ESBL-harboring plasmids in broilers, while the effect on horizontal gene transfer is less obvious.

Prevalence of AmpC and Extended-Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae and Escherichia coli Isolates

Background: In recent years, the prevalence of antibiotic resistance has steadily increased and also the antibiotic-resistant strains producing extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases have emerged among the Enterobacteriaceae, predominantly in Escherichia coli and Klebsiella pneumoniae species. Objectives: This prospective study aimed at determining the production of ESBL or AmpC, phenotypically and also at the molecular level, in E. coli and K. pneumoniae isolates collected from various clinical specimens. Methods: In total, 78 K. pneumoniae and 92 E. coli isolates were collected from various clinical infectious sources available in different wards of the Imam Reza Hospital, Tabriz, Iran, from July 2017 to December 2018. All isolates were subjected to antimicrobial susceptibility testing. MAST 4-disc test and polymerase chain reaction (PCR) were applied for phenotypic and genotypic detection of ESBLs and plasmid-encoded AmpCs (pAmpCs) among isolates, respectively. Results: Overall, 78 K. pneumoniae and 92 E. coli isolates were evaluated, of which 46 K. pneumoniae (58.9%) and 51 E. coli (55.4%) isolates were resistant to cefotaxime/ceftazidime and included in the study. Among the K. pneumoniae and E. coli isolates resistant to cefotaxime/ceftazidime, 40 (86.9%) and 40 (78.4%) isolates were ESBL producers and 8 (17.3%) and 2 (3.9%) isolates were pAmpC producers, respectively. In addition, 40 E. coli (78.4%) isolates were positive for both CTX-M-14 and CTX-M-15 genes. Regarding K. pneumoniae isolates, 40 isolates (86.9%) were positive for CTX-M-15 gene and 18 isolates (39.1%) for CTX-M-14 gene. Among 51 ceftazidime/cefotaxime-resistant E. coli isolates, 32 isolates (62.7%) were positive for DHA-1 gene and 33 isolates (64.7%) isolates for CMY-2 gene. Also, among 46 ceftazidime/cefotaxime-resistant K. pneumoniae isolates, 15 isolates (32.6%) had DHA-1 gene and 27 isolates (58.7%) had CMY-2 gene in the genome. Conclusions: The high prevalence of ESBL and AmpC production among E. coil and K. pneumoniae isolates was a serious concern in the studied region. Therefore, a simple and rapid PCR-based technique is essential to detect and distinguish various pAmpC and ESBL genes to discriminate other resistance determinants

Multidrug-Resistance Genes in Pseudomonas aeruginosa from Wound Infections in a Tertiary Health Institution in Osogbo, Nigeria.

Multidrug Resistant Pseudomonas aeruginosa (MDRPA) is a ubiquitous opportunistic organism that poses threat to the management of infections globally. The objectives of the current research were to assess the antibiotic resistance profiles as well as Multiple Antibiotic Resistance (MAR) Index of clinical isolates of P. aeruginosa associated with wound infections. Presence of Extended Spectrum Beta Lactamase genes (bla CTX-M, bla SHV and bla TEM) and Carbapenemase genes (bla KPC and blaNDM) were also determined among the isolates. Swab samples were collected from 255 patients with wound infections. Bacterial identification was done by standard diagnostic tests. The identity of isolates was confirmed by the detection of the exoA gene using the PCR technique. Antibiotic susceptibility testing and resistance profile were determined using the disc diffusion method. Resistance genes were amplified by the PCR method. A total of 235 (92.2%) bacterial isolates were recovered from the wounds of the 255 patients, of these, 124 (52.8%) were Gram-negative bacilli while the remaining 111 (47.2%) were Gram-positive cocci. A total of 69 Pseudomonas aeruginosa strains were recovered from the wound specimens. Imipenem was the most effective antibiotic against these isolates (92.8% isolates were susceptible) while all isolates were resistant to Meropenem, Cefepime, Ticarcillin, Amoxicillin-clavulanic acid, Cefotaxime, Ampicillin and Cefpodoxime. All 69 Pseudomonas aeruginosa isolates were multidrug resistant (MDR). Of the isolates selected for PCR, all were positive for TEM, CTX-M and SHV genes while one-third were blaKPC and blaNDM producers. This study demonstrated high prevalence of carbapenem-resistant strains of P. aeruginosa, suggesting that there is an urgent need in Nigeria for the enactment and enforcement of policies and necessary laws restricting the availability and indiscriminate use of antibiotics.

Genomic surveillance for hypervirulence and multi-drug resistance in invasive Klebsiella pneumoniae from South and Southeast Asia

BackgroundKlebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired “hypervirulent” strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide.MethodsWe conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate, a K. pneumoniae-specific genomic typing tool.ResultsK. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common among South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes among K. pneumoniae.ConclusionsK. pneumoniae BSI in South and Southeast Asia are caused by different STs from those predominating in other regions, and with higher frequency of acquired virulence determinants. K. pneumoniae carrying both iuc and AMR genes were also detected at higher rates than have been reported elsewhere. The study demonstrates how genomics-based surveillance—reporting full molecular profiles including STs, AMR, virulence and serotype locus information—can help standardise comparisons between sites and identify regional differences in pathogen populations.

High Prevalence of CTX-M Type Extended-Spectrum Beta-Lactamase Genes and Detection of NDM-1 Carbapenemase Gene in Extraintestinal Pathogenic Escherichia coli in Cuba.

Increase of extraintestinal pathogenic Escherichia coli (ExPEC) showing resistance to beta-lactams is a major public health concern. This study was conducted as a first molecular epidemiological study on ExPEC in Cuba, regarding prevalence of extended-spectrum beta-lactamases (ESBLs) and carbapenemase genes. A total of 306 ExPEC isolates collected in medical institutions in 16 regions in Cuba (2014–2018) were analyzed for their genotypes and presence of genes encoding ESBL, carbapenemase, plasmid-mediated quinolone resistance (PMQR) determinants by PCR and sequencing. The most common phylogenetic group of ExPEC was B2 (49%), followed by D (23%), A (21%), and B1 (7%). Among ESBL genes detected, blaCTX-M was the most common and detected in 61% of ExPEC, with blaCTX-M-15 being dominant and distributed to all the phylogenetic groups. NDM-1 type carbapenemase gene was identified in two isolates of phylogenetic group B1-ST448. Phylogenetic group B2 ExPEC belonged to mostly ST131 (or its single-locus variant) with O25b allele, harboring blaCTX-M-27, and included an isolate of emerging type ST1193. aac (6’)-Ib-cr was the most prevalent PMQR gene (40.5%), being present in 54.5% of CTX-M-positive isolates. These results indicated high prevalence of CTX-M genes and the emergence of NDM-1 gene among recent ExPEC in Cuba, depicting an alarming situation.

Klinik E.coli İzolatlarında Virulans Faktor Genlerinin ve Geniş Spekturumlu Beta Laktamazların Araştırılması

The aim of this study was to investigate the presence of pap, hly, cnf, sfa, aer and afa virulans genes and extended-spectrum beta lactamase resistance genes in 83 clinical E.coli isolates. In 83 E. coli isolates, sfa, pap, hly, cnf, aer and afa virulence genes and TEM-SHV, CTX-M1 and CTX-M9 wide spectrum beta-lactamase genes were investigated by polymerase chain reaction (PCR) method. Resistance rates of 83 isolates to cefixim, trimethoprim/sulfamethoxazole, piperacillin/tazobactam, amoxicillin/clavulanic acid, ampicillin, ceftriaxone, cefuroxime, cefuroxime axetil, ciprofloxacin, fosfomycin, gentamicin and ceftazidime antibiotics were 65%, 54%, 19%,38, 74%, 75%, 72%, 75%, 68%, 2%, 30%, 49%, respectively. CTX-M1 was the most common extended spectrum beta-lactamase (ESBL) among the ESBL genes investigated in 51 of 83 strains. TEM type beta-lactamase was detected in 33 strains, CTX-M9 and SHV 1 isolates. The most common virulence gene was aer gene and detected in 40 strains, followed by the cnf gene detected in 15 of the isolates. The pap, hly, afa and sfa genes were determined in 15.6%, 12.04%, 9.6% and 15.6% of the isolates, respectively. In addition, 10 different virulence factor gene patterns were observed in 83 E.coli isolates. Virulence gene combinations were observed in 27 strains of aer, 6 strains of cnf-sfa-pap, 1 strains of aer-pap, 2 strains of aer-afa, 6 strains of afa, 3 strain of sfa, 9 strain of aer-cnf-hly, 4 isalates of sfa-pap, 1 strain of hly-pap and 1 strains of pap genes. In conclusion, investigating bacterial pathogenicity associated with UTI will contribute to better medical intervention.

Rapid Detection of Genes Encoding Extended-Spectrum Beta-Lactamase and Carbapenemase in Clinical Escherichia coli Isolates with eazyplex SuperBug CRE System.

Objectives: This study evaluated the diagnostic performance of the eazyplex® SuperBug CRE (eSBCRE) system, based on a loop-mediated isothermal amplification (LAMP), for the detection of the most common extended-spectrum beta-lactamases (ESBL) and carbapenemase genes in 140 clinical isolates of Escherichia coli. Materials and Methods: ESBL (blaCTX-M-1group and blaCTX-M-9group) and carbapenemase (blaKPC, blaVIM, blaNDM, blaOXA-48, and blaOXA-181) genes were detected using the eSBCRE test and compared with the results obtained by PCR, real-time PCR, and phenotypic methods. Results: Concordant results of 100% between PCR/real-time PCR and eSBCRE assays were observed. Two of 140 E. coli isolates were positive for both ESBL and carbapenemase genes according to eSBCRE, PCR, and real-time PCR assays, whereas they were negative in double-disk synergy test. Of 16 E. coli isolates suspected of producing carbapenemase, 9 were positive for 48-oxacillinases (OXA-48) by 30 μg temocillin test, whereas the blaOXA-48 was found only in 1 E. coli isolate by all molecular methods. Maximum threshold time values (minutes:seconds) in the eSBCRE test were 6:00, 11:15, 11:00, and 9:00 for the blaCTX-M-1group, blaCTX-M-9group, blaVIM, and blaOXA-48 genes, respectively. Conclusions: The eSBCRE test based on LAMP method is a reliable, easy-to-use, and timesaving molecular system, which can be successfully used in the routine diagnostic for the rapid detection of the most common ESBL and carbapenemase genes among clinical E. coli isolates.

The Inhibitory Effects of Lactobacillus Supernatants and Their Metabolites on the Growth and Biofilm Formation of Klebsiella pneumoniae.

Klebsiella pneumoniae is a common cause of hospital-acquired infections, including urinary tract infection (UTI). Biofilm formation makes the K. pneumoniae infection more complicated and carrying extended-spectrum beta-lactamases (ESBLs) genes, it limits antibiotic choices for treatment. Lactobacillus strains are known as natural protective barriers against UTIs. This is a small in-vitro study aimed to determine the effect of probiotic Lactobacillus strains and some types of their metabolites on the growth and biofilm of UTI isolates and reference strain of Klebsiella pneumoniae. The efficacy of Lactobacillus supernatants and antibiotics in the prevention and elimination of K. pneumoniae biofilms was determined using a quantitative adherence assay. A rapid colorimetric microplate bioassay was applied for the detection of survived bacterial cells after treatment with antibacterial agents. Biofilm phenotypes were studied by scanning electron microscopy (SEM). The results showed that seven out of eight ESBL producing uropathogenic K. pneumoniae isolates in this study were able to produce biofilm. Lactobacillus supernatants at 1:1 to 1:16 dilutions, had more than 95% biofilm-inhibitory and biofilm-killing properties on a strong biofilm producer isolate. Supra-MIC levels of antibiotics had a much lower anti-biofilm effect than Lactobacillus supernatant and left considerable alive biofilm cells. Although antibiotic resistance increases in biofilm forms of Klebsiella pneumoniae, Lactobacillus supernatants have strong antibiofilm efficacy even in lower concentrations of MIC. Biofilm formation decreases considerably in the presence of Lactobacillus supernatants. Hydrogen peroxide is an effective product against growth and biofilm formation of Klebsiella pneumoniae.