Enterobacterial Repetitive Intergenic Consensus PCR Research Articles

Occurrence, Antibiotic Resistance, Virulence Factors, and Genetic Diversity of Bacillus spp. from Public Hospital Environments in South Africa.

This study aimed to assess the molecular dissemination of Bacillus species in public hospitals in South Africa. The study conducted over 3 months during 2017 involved representative samples obtained from three wards (general ward, intensive care unit, and pediatric unit) from four public hospitals denoted as A (Central), B (Tertiary), C (Regional), and D (District). Swabs collected from 11 distinct hospital surfaces were screened using selective media, biochemical testing, and molecular methods. Overall, 17% (135/777) isolates were identified with a prevalence of 24% (32/135) for central, 33% (45/135) for tertiary, 27% (36/135) for regional, and 16% (22/135) for district hospital. Bacillus species were further confirmed to belong to Bacillus cereus (129/135; 96%) and Bacillus subtilis (6/135; 4%). Prevalence was similar across the wards, averaging 33.3% (45/135). The highest prevalence of Bacillus isolates was found on the drip stands (11.8%), sink (11.8%), ward phone (11.5%), and nurses' tables (10.3%). Minimum inhibitory concentration analyses revealed high resistance to β-lactams, fluoroquinolones, and tetracyclines. The most common resistance genes detected were ermB (56%) and tetM (5%). Enterotoxin virulence genes hblA (77%) and hblD (88%) associated with the diarrheal syndrome were most detected; however, no ces genes (cereulide toxin) for emetic syndrome was found. The enterobacterial repetitive intergenic consensus PCR revealed considerable diversity at the different levels of health care, although the clonal spread of strains between the sites/wards within each specific hospital was revealed. The study highlighted the dissemination of drug-resistant Bacillus spp. in public hospital environments and calls for the design of optimal strategies to curb their spread.

Biotyping of isolates of Pseudomonas aeruginosa isolated from human infections by RAPD and ERIC-PCR

Pseudomonas aeruginosa is a significant mortality factor due to nosocomial infections in humans. P. aeruginosa has been known with severe infections, high incidence, and multiple drug resistance. The present study aims to rapidly diagnose and biotype the isolates of P. aeruginosa isolated from human infections in Shiraz hospitals and health centers. Ninety six different isolates were collected from skin, urine, sputum, blood, wound, central vein blood, body fluids and burn wounds between January 2016 and February 2017. After phenotypic confirmation, isolates were examined by PCR for molecular confirmation. Ninety three isolates were verified as P. aeruginosa in molecular analysis. Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and Random Amplified Polymorphic DNA (RAPD) were done for 67 isolates. In ERIC-PCR, the patterns obtained included 2–11 bands. The RAPD patterns obtained with primers 272 and 208 consisted of 3–11 and 1–12 bands respectively. Based on dice similarity coefficient of greater than 80%, 38, 45 and 38 groups were identified in ERIC, RAPD 272 and RAPD 208 respectively. The results showed that the isolates of P. aeruginosa have a high polymorphism apparently because of the high genetic variation.

Biofilm formation by uropathogenic Escherichia coli: a complicating factor for treatment and recurrence of urinary tract infections

Uropathogenic Escherichia coli (UPEC) are the principal cause of urinary tract infections (UTIs) which can be either hospital- or community-acquired. The most crucial factor in the persistence and recurrence of UTIs is the biofilm formation ability of UPEC, which protects them against antimicrobial treatment. To investigate the genetic relatedness, biofilm formation ability, and biofilm-related genes in UPEC isolated from hospital- and community-acquired UTI patients. Invitro biofilm formation ability of 100 UPEC isolates, collected from the urine samples of 49 inpatients and 51 outpatients with UTIs, was assessed by the microtitre plate method. The association between the presence of fimH, papC, sfa/focDE, csgA, crl, afa, flu, and bcsA genes and biofilm formation ability of UPEC was statistically analysed. The genetic relatedness of UPEC isolates was evaluated by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Overall, 99% of the UPEC isolates showed invitro biofilm formation ability, and 27% of the isolates were moderate to strong biofilm producers. Only the presence of sfa/focDE gene was significantly associated with moderate and strong biofilm formation by the UPEC isolates. Analysis of dendrograms revealed higher genetic similarities among UPEC isolates of inpatients compared with outpatients. Based on the results, selection of effective therapeutic approaches, which can affect both biofilm formation and enclosed UPEC, is important for preventing recurrent UTIs. The common UPEC clones among inpatients in different hospital units emphasize the need for more rigid control measures to prevent the spread of UPEC in hospitalized patients and to reduce the occurrence of hospital-acquired UTIs.

Antibiotics Combinations and Chitosan Nanoparticles for Combating Multidrug Resistance Acinetobacter baumannii.

BackgroundSuccessful treatment of Acinetobacter (A.) baumannii-associated infection is complicated by the emergence of multidrug resistance (MDR), particularly in clinical settings. This urges searching for new alternatives to encounter such health problem.AimThis study aimed to evaluate certain antibiotic combinations and CNPs either alone or in combination of some selected antibiotics for the purpose of combating MDR A. baumannii clinical isolates.MethodsA total of 51 A. baumannii clinical isolates were recovered from discharged clinical specimens of the Clinical Microbiology Central Laboratory of AL Kasr Al Aini hospital, Cairo, Egypt. Conventional standard Lab tests were used for identification followed by recA gene testing for confirmation. Antimicrobial susceptibility tests were conducted out according to CLSI guidelines. Genotypic analysis using Enterobacterial Repetitive Intergenic Consensus-polymerase chain reaction (ERIC-PCR) of the respective isolates showed that they were clustered in nine clones. The prepared CNPs were characterized by dynamic light scattering and HR-transmission electron microscope imaging. Antibiotic combinations and co-effect of CNPs with some selected antibiotics (either each alone or in combination of two) were evaluated using the Checkerboard microdilution and minimum inhibitor concentration decrease factor (MDF) methods, respectively.ResultsThe recovered 51 A. baumannii clinical isolates were MDR (100%) of these 92% (47/51) were extensively drug resistance (XDR). Combinations of colistin (CT)+meropenem (MEM) and MEM+tigecycline (TGC) showed synergism in 77.7% and 44.4% and additive effects in 22.3% and 55.6% of the tested MDR A. baumannii isolates (n=51), respectively. However, CT+TGC combination showed antagonism. CNPs exhibited good inhibitory activity (inhibition zones ranged from 24 to 31 mm) against selected nine MDR A. baumannii isolates (one isolate from each clone). The MIC of CNPs at concentrations (ranging from 1 to 5 mg/mL) were from 0.16 to 0.25 mg/mL, indicating good in vitro antimicrobial activities. CNPs (5 mg/mL) when combined with CT, TGC or MEM, CT+MEM and TGC+MEM significantly increased the susceptibilities of the MDR A. baumannii isolates to these antibiotics by 88.8%, 66.6%, 100%, 77.7%, and 44.4%, respectively. No significant effects were observed when CNPs (5 mg/mL) were combined with CT+TGC.ConclusionThe current study demonstrated the significant in-vitro activities of CNPs either alone or in combination with CT, TGC or MEM, CT+MEM and TGC+MEM and the successful combinations of MEM either with CT or with TGC against the MDR A. baumannii pathogens. However, further in vivo studies should be conducted to verify such activities and their potential use in human.

Prevalence and Characteristics of Metallo-beta-Lactamase-positive and High-risk Clone ST235 Pseudomonas aeruginosa at Ardabil Hospitals

Background: Carbapenems are the most commonly administered drugs for the treatment of multidrug-resistant Pseudomonas aeruginosa (MDR P. aeruginosa) infections. However, carbapenem-resistant P. aeruginosa is spreading rapidly and has led to a new threat to human health worldwide. Objectives: The current study aimed to determine the prevalence of imipenem-resistant P. aeruginosa, detect metallo-β-lactamase (MBL)-producer isolates, and evaluate their clonal relationships in strains isolated from patients referring to the hospitals of Ardabil city, Iran. Methods: The resistance rate to imipenem was evaluated using the disk diffusion method. Double-disk synergy test and PCR technique were used for phenotypic and genotypic screening of MBL-positive P. aeruginosa, respectively. Ultimately, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequence typing (MLST) methods were used for assessing clonal relatedness among the isolates. Results: The prevalence of imipenem-resistant P. aeruginosa strains was estimated at 57.1% (48 out of 84 isolates). In addition, 45 (93.7%) out of 48 imipenem-resistant P. aeruginosa isolates were phenotypically screened as MBL-positive, among which 16 (35.5%) and three (6.6%) isolates harbored blaIMP and blaVIM-1 genes, respectively. However, blaNDM, blaSIM-2, blaSPM, and blaGIM-1 genes were not detected in this study. MBL-producing P. aeruginosa strains were divided into 42 ERIC-PCR types. Based on the results of MLST, P. aeruginosa ST235 was the only identified sequence type. Conclusions: Our results revealed a high and alarming prevalence of imipenem-resistant and blaIMP-positive P. aeruginosa ST235 at Ardabil hospitals. Continuous monitoring is essential to control the further spread of this highly virulent and drug-resistant clone.

Molecular Epidemiology of Multidrug-resistant Escherichia coli from Urinary Tract Infections

Objectives: The purpose of this study was to investigate the phylogenetic groups, antibiotic resistance, antibiotic resistance genes (ARGs), integrons, extraintestinal virulence genes and genetic diversity of Escherichia coli isolates from human urinary tract infection. Methods: A total of 100 E. coli isolates were collected from patients with urinary tract infections in Kerala, South India. Antibiotic susceptibility testing of all E. coli isolates against different antibiotics was determined by the disc diffusion method. Phylogenetic groups, extraintestinal virulence genes, ARGs, and integrons were detected by PCR. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was used to check the genetic relatedness among E. coli isolates. Results: E. coli isolates have mainly belonged to phylogenetic group B2. Resistance to ampicillin was most frequent among the E. coli isolates followed by resistance to cefoxitin, cefpodoxime, nalidixic acid, trimethoprim, and co-trimoxazole. Among E. coli isolates, 96% were multidrug-resistant (MDR), and 86% and 32% harbored ARGs and integrase 1 (int1) respectively. Seventy-nine percent of the isolates were extraintestinal pathogenic E. coli (ExPEC), and 86% of them (n = 68) harbored ARGs. One extensively drug-resistant (XDR) ExPEC was obtained in this study. The present study revealed a significant association between the presence of virulence genes and antibiotic resistance. A high degree of genetic diversity was observed among the ARGs-harboring E. coli isolates. Conclusion: Understanding the association between extraintestinal virulence genes and antibiotic resistance genes would result in the proper treatment of urinary tract infections. J Microbiol Infect Dis 2021; 11(2):66-73.

Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan.

Chinese softshell turtles (Pelodiscus sinensis) (CST) are susceptible to infections by bacteria belonging to the Bacillus cereus group (Bcg). Bcg includes several closely related species, two of which, B. cereus and B. thuringiensis, are pathogens of aquatic animals or insects. In the present study, we collected 57 Bcg isolates obtained from diseased CST from 2016 to 2019 in Kaohsiung and Pingtung, the areas with the most CST farms in Taiwan. All isolates were divided into four genotypes with two restriction enzymes, SmaI and NotI, by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Representative isolates from each genotype were subjected to phylogenetic tree analysis using 16S rDNA and pyruvate carboxylase genes as phylogenetic markers, and these CST isolates appeared in different clades. PCR was performed targeting six selected virulence genes, four of which were detected in CST isolates, including cytotoxin K (1/57), hblC of the haemolysin BL complex (46/57), nheA of the non-haemolytic enterotoxin complex (52/57) and enterotoxin FM (57/57), whereas cereulide synthetase and cereulide peptide synthase-like genes were not detected in any isolates.

Molecular epidemiology of vancomycin-resistant Enterococcus faecium clinical isolates in a tertiary care hospital in southern Thailand: a retrospective study

ObjectiveVancomycin-resistant enterococci are nosocomial pathogens that are responsible for commonly causing healthcare-associated infections, and they exhibit increased resistance to many antimicrobials, particularly to vancomycin. The epidemiological data available on vancomycin-resistant enterococci (VRE) in Thailand are inadequate.MethodsUsing enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), this study investigated genes that encode antimicrobial resistance and genetic relatedness to further understand VRE prevalence. Ninety VRE isolates were collected between 2011 and 2019 from a tertiary care hospital in southern Thailand. Antimicrobial susceptibility was determined using the disk diffusion method and E-test methods. Multiplex PCR was performed to detect the van gene and virulence genes.ResultsThe study showed a high prevalence of diverse multidrug-resistant VRE strains. The prevalence of VRE infection was the highest in 2014 (28 isolates, 39.4%). VRE were mostly found in the urogenital tract (26 isolates, 28.9%), followed by the digestive tract (20%), body fluid, i.e., pancreatic cyst fluid, peritoneal dialysis fluid, Jackson–Pratt (JP) drain (20%), and blood specimens (10%). Patients in medical and surgical wards had 71.1% multi-drug-resistant and 28.9% extensively drug-resistant (XDR) VRE strains, respectively. The most prevalent antibiotic resistance was to ampicillin (74.4%). Susceptibility to gentamicin and meropenem were similar (7% and 10%, respectively). Four isolates (4.4%) were resistant to colistin. Only vanA was detected among the strains. The virulence gene test showed that the detection rates of enterococcal surface protein (esp) and hyaluronidase (hyl) genes were 91.1% and 5.6%, respectively. According to ERIC-PCR analysis, 51 of 90 strains had clonality, with a similarity rate of 95%.ConclusionsWe conclude that there is a need to implement infection control practices and active surveillance. Molecular techniques can effectively detect antibiotic-resistant genes, which would allow monitoring to control VRE infection in hospitals.

Which missense mutations associated with DNA gyrase and topoisomerase IV are involved in Pseudomonas aeruginosa clinical isolates resistance to ciprofloxacin in Ardabil?

Ciprofloxacin is one of the most effective antibiotics against Pseudomonas aeruginosa (P. aeruginosa) infections. However, the prevalence of ciprofloxacin-resistant P. aeruginosa isolates is increasing in the world. In the current study, we assessed ciprofloxacin resistance rate, the mechanism of resistance to ciprofloxacin and also clonal relationships among ciprofloxacin-resistant P. aeruginosa in Ardabil. Applying phenotypic and genotypic methods, a total of 84 isolates of P. aeruginosa were collected from clinical specimens in Ardabil hospitals. P. aeruginosa resistance to ciprofloxacin antibiotic was evaluated using disk diffusion test. Genomic DNA was extracted using boiling method. Amplification of the gyrA, gyrB, parC and parE genes and analysis of gene mutations and amino acid alterations were performed through polymerase chain reaction (PCR) and sequencing assays. Finally, genetic diversity of ciprofloxacin-resistant P. aeruginosa isolates was determined by the enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) typing method. P. aeruginosa resistance to ciprofloxacin was 48.8%. The most prevalent missense mutations in the QRDR regions of target genes were as follows: Thr83Ile and Asp87Asn in the GyrA subunit, and Ser87Leu and Ser87Trp in the ParC subunit. There were no amino acid alterations in the GyrB and ParE subunits in ciprofloxacin-resistant P. aeruginosa isolates. In addition, ciprofloxacin-resistant P. aeruginosa were divided into 34 ERIC-PCR types, out of which type 27 was the most prevalent genotype. P. aeruginosa resistance to ciprofloxacin was high in our study and missense mutations were involved in ciprofloxacin resistance. Hence, continuous monitoring of ciprofloxacin resistance along with genotyping of resistant isolates and identification of other mechanisms of resistance are recommended for a better management of P. aeruginosa infections.

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli strains in dairy farm wastewater in Chiang Mai

We investigated the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains in dairy farm wastewater in Chiang Mai, Thailand. We analyzed wastewater samples collected from 150 dairy farms and found that 88.7% of the farms (n = 133) were positive for ESBL-producing E. coli. Multiplex polymerase chain reaction (PCR) amplification was performed to characterize the presence of bla CTX-M, bla TEM, and blaSHV in ESBL-producing isolates. blaCTX-M was found in all isolates (n = 133), followed by blaTEM (80/133, 60.2%), whereas blaSHV was not detected in any isolate. blaCTX-M and blaTEM were present in 60.2% (80/133) of the isolates, and 39.8% (53/133) isolates carried bla CTX-M alone. Subgroup analysis showed that CTX-M-1 was the most prevalent subgroup among the isolates (129/133, 97.0%), followed by CTX-M-8 (2/133, 1.5%) and CTX-M-9 (2/133, 1.5%). The distribution of the phylogenetic groups was as follows: group A (100/133, 75.2%), followed by B1 (14/133, 10.5%), D (6/133, 4.5%), F (6/133, 4.5%), B2 (4/133, 3.0%), and E (3/133, 2.3%). Based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and dendrogram analysis, 24 isolates were classified into clades I (n = 21), II (n =1), and III (n =2). Minor genetic differences were found in all clade I isolates. Our data suggest that the circulating of ESBL-producing E. coli carried at least one bla gene strain distributed in dairy farm wastewater in Chiang Mai.

Prevalence, Genetic Diversity, Antimicrobial Resistance, and Toxigenic Profile of Vibrio vulnificus Isolated from Aquatic Environments in Taiwan.

Vibrio vulnificus is a gram-negative, opportunistic human pathogen associated with life-threatening wound infections and is commonly found in warm coastal marine water environments, globally. In this study, two fishing harbors and three tributaries of the river basin were analyzed for the prevalence of V. vulnificus in the water bodies and shellfish that are under the pressure of external pollutions. The average detection rate of V. vulnificus in the river basins and fishing harbors was 8.3% and 4.2%, respectively, in all seasons. A total of nine strains of V. vulnificus were isolated in pure cultures from 160 samples belonging to river basins and fishing harbors to analyze the antibiotic susceptibility, virulence gene profiles, and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) fingerprinting. All isolates were susceptible to 10 tested antibiotics. The genotypic characterization revealed that 11.1% (n = 1/9) strain was nonvirulent, whereas 88.9% (n = 8/9) isolates were virulent strains, which possessed the four most prevalent toxin genes such as vcgC (88.9%), 16S B (88.9%), vvhA (88.9%), and manIIA (88.9%), followed by nanA (77.8%), CPS1 (66.7), and PRXII (44.4%). Additionally, ERIC-PCR fingerprinting grouped these nine isolates into two main clusters, among which the river basin isolates showed genetically diverse profiles, suggesting multiple sources of V. vulnificus. Ultimately, this study highlighted the virulent strains of V. vulnificus in the coastal aquatic environments of Taiwan, harboring a potential risk of infection to human health through water-borne transmission.

Serotyping and Antimicrobial Resistance Profile of Enteric Nontyphoidal Salmonella Recovered from Febrile Neutropenic Patients and Poultry in Egypt.

A total of 300 human fecal samples were collected from febrile neutropenic patients suffering from severe gastroenteritis, followed by identification and serological characterization of recovered isolates. Fifty nontyphoidal Salmonella (NTS) serovars were recovered. A total of serologically identified 50 NTS serovars recovered from poultry of the same geographical area and during the same period as well as one standard strain S. Poona were supplied by the Bacterial Bank of Animal Health Research Institute of Egypt. Antibiogram analysis revealed that the human and poultry serovars exhibited similar antimicrobial resistance patterns against 28 different antimicrobial agents, particularly against ampicillin, cefotaxime, oxytetracycline, and erythromycin. Plasmids harboring blaCTX-m, blaSHV, blaTEM, and aac(6’)-Ib were detected in 11 (22%) and 8 (16%) of human and poultry serovars, respectively. Molecular detection of the most clinically relevant virulence genes and analysis of the associated virulence genotypes proved that the human (n = 11) and poultry serovars (n = 12) shared 11 genotypes. Enterobacterial repetitive intergenic consensus PCR analysis revealed that human and poultry serovars were clustered together in 3 out of the 4 clusters with a similarity index ranged from 0.15 to 1. Since poultry are usually consumed by humans, the presence of resistant bacteria harboring transmissible genetic elements is of great health concern.

Antibiotic resistance and determination of resistant genes among cockle (Anadara granosa) isolates of Vibrio alginolyticus.

A total of 24 strains of Vibrio alginolyticus were isolated from cockles (Anadara granosa) and identified for VibA and gyrB genes. All V. alginolyticus isolates were then tested against nine different antibiotics. In this study, the highest percentage of antibiotic resistance was obtained against penicillin (37.50%), followed by ampicillin, vancomycin (12.50%) and erythromycin (8.33%). All of V. alginolyticus isolates were susceptible against streptomycin, kanamycin, tetracycline, chloramphenicol and sulfamethoxazole. Polymerase chain reaction (PCR) assay has confirmed the presence of four antibiotic resistance genes of penicillin (pbp2a), ampicillin (blaOXA), erythromycin (ermB) and vancomycin (vanB). Out of 24 V. alginolyticus isolates, 2 isolates possessed the tdh-related hemolysin (trh) (strains VA15 and VA16) and none for the thermostable direct hemolysin (tdh) gene. Both strains of the tdh-related hemolysin (trh) were susceptible to all antibiotics tested. The multiple antibiotic resistance (MAR) index ranging between 0.2 and 0.3 with 5 antibiograms (A1-A5) was observed. Combination of enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and antibiotic resistance indicated 18 genome types which showed genetic heterogeneity of those V. alginolyticus isolates. The results demonstrated the presence of V. alginolyticus strain found in cockles can be a potential risk to consumers and can contribute to the deterioration of human health in the study area. Thus, it is essential for local authority to provide the preventive measures in ensuring the cockles are safefor consumption.

Phenotype-Genotype Characterization and Antibiotic-Resistance Correlations Among Colonizing and Infectious Methicillin-Resistant Staphylococcus aureus Recovered from Intensive Care Units.

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) presents a profound hazard to public health. MRSA colonizing skin, mucous membranes, and the anterior nares without clinical symptoms is termed “colonizing MRSA”. Upon manifestation of clinical symptoms, it is termed “infectious MRSA”. Here, we characterize and differentiate colonizing and infectious MRSA, and analyze the phenotypic-genotypic and antibiotic susceptibility correlations.MethodologyClinical MRSA isolates were recovered from intensive care units (ICUs) of two major Egyptian hospitals and their biofilm formation ability was tested. Antibiograms against 16 antibiotics were determined, in addition to the minimum inhibitory concentrations (MICs) of vancomycin and linezolid. The entire collection was typed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as multi-locus sequence typing (MLST). Representative resistance and virulence genes were detected by PCR amplification.ResultsForty-nine isolates were confirmed as MRSA, of which 30 isolates were infectious and 19 were colonizing. Versatile resistance patterns were observed in both groups of isolates. We report a higher tendency for biofilm-formation and borderline minimum inhibitory concentrations among infectious isolates. A Positive antibiotic correlation was observed between susceptibility to protein synthesis inhibitors and cell wall inhibitors. Positive correlations were observed between isolation site and rifampicin resistance: nasal samples were enriched in rifampicin-resistant isolates, while urine and blood samples were enriched in susceptible ones. Furthermore, biofilm formation ability was slightly associated with amikacin resistance, and an association between teicoplanin resistance and the presence of the Panton-Valentine leukocidin gene was the only significant phenotype-genotype correlation observed. Finally, ERIC typing and MLST had congruent results.ConclusionLinezolid and vancomycin are still the most convenient choice for MRSA treatment. ERIC PCR and MLST show promising typing combination that could be easily used periodically for tracking the genotypic changes of MRSA, especially within the healthcare facilities. Several correlations were established between groups of antibiotics and the genotypes/phenotypes of the selected isolates.

Distribution, contamination routes, and seasonal influence of persistent Listeria monocytogenes in a commercial fresh Hypsizigus marmoreus production facility

Listeria monocytogenes is an important foodborne pathogen worldwide, with a high mortality rate of 20%–30% for vulnerable persons. The aim of this study was to determine the distribution, contamination routes, and persistence of L. monocytogenes in a mushroom plant. A total of 204 samples collected during four sampling periods within a one-year time interval, with 27.0% (55/204) were positive for L. monocytogenes. A total of 60 representative L. monocytogenes strains were selected for further analysis based on serogrouping and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) fingerprints. Multiplex PCR showed serogroups I.1 (1/2a-3a) and II.2 (1/2b-3b-7) accounted for 68.3% (41/60) and 31.7% (19/60), respectively, of the samples. ST8 strains were isolated from the mycelium-scraping phase to the harvest phase by multilocus sequence typing. These strains had identical ERIC-PCR fingerprints, suggesting that the mycelium-scraping machine was the main contamination source. Sequence types (ST) 8 (61.7%, 37/60) and 87 (23.3%, 14/60) were predominant and repeatedly isolated within a one-year seasonal investigation, indicating that ST8 and ST87 persist in this plant. The minimum inhibitory concentrations of commonly used quaternary ammonium compounds for the 60 isolates were ≥4 μg/mL, indicating that the persistence we observed may be associated with biocide tolerance. In addition, all strains harbored nine virulent genes (prfA, hly, mpl, actA, iap, plcA, plcB, inlA, and inlB). Only ST87 strains carried ptsA (representing LIPI-4), but none of the strains harbored llsX (representing LIPI-3). These results indicate that ST87 strains pose a potential public risk to consumers. All isolates were susceptible to 13 antimicrobials, except for levofloxacin (1.7%, 1/60), erythromycin (3.3%, 2/60), and clindamycin (88.3%, 53/60). This study is the first to explore the contamination routes and persistence of L. monocytogenes in a Hypsizigus marmoreus production facility. Critical disinfection measures should be adopted to eliminate its persistence throughout processing.

Characterization of antibiotic resistance profiles in Pseudomonas aeruginosa isolates from burn patients

ObjectiveTo investigate the prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) producing extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria. MethodsBetween April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR). ResultsAmong the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for six groups of bla genes encoding ESBL-type enzymes, namely blaCTX-M2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Out of the 28 ESBL-producing strains, 23 (82.14%) were blaCTX-M2 positive; 18 (38.29%) were blaPER positive, and 16 (34.04%) were blaTEM positive, while 5 (17.9%) were co-harboring blaCTX-M2, blaTEM, and blaPER genes. The blaSHV, blaVEB, and blaGES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the blaNDM-1, blaIMP, blaVIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the oprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 11 distinct PA clones among the blaCTX-M2 positive strains. ConclusionThis is the first study to report the presence of CTX-M2-producing PA in the North Africa region and the first to detect blaCTX-M2-positive and blaPER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains to this part of the world. Identification of bacterial genotypic alterations that confer antibiotic resistance is critical in determining the most effective antimicrobial strategies to be employed. Therefore, our findings could potentially facilitate clinical decision making regarding the antibiotics of choice for the treatment of burn patients that suffer from PA infections in Algeria.

Extensively Drug-Resistant Hypervirulent Klebsiella pneumoniae From a Series of Neonatal Sepsis in a Tertiary Care Hospital, India.

The recent emergence of multidrug-resistant (MDR) Klebsiella pneumoniae with hypervirulent traits causing severe infections and considerable mortality is a global cause for concern. The challenges posed by these hypermucoviscous strains of K. pneumoniae with regard to their optimal treatment, management, and control policies are yet to be answered. We studied a series of extensively drug-resistant (XDR) and hypervirulent K. pneumoniae ST5235 isolates with resistance to carbapenems and polymyxins causing neonatal sepsis in a tertiary care hospital in India. A total of 9 K. pneumoniae isolates from 9 cases of neonatal sepsis were studied with respect to their clinical relevance, antimicrobial susceptibility profile, presence of extended spectrum β lactamase (ESBL) production, and responsible genes, carbapenemases (classes A, B, and D), and aminoglycoside-resistant genes. Hypervirulence genes encoding hypermucoid nature, iron uptake, and siderophores were detected by multiplex PCR. The plasmid profile was studied by replicon typing. Isolates were typed by multilocus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC) PCR to study the sequence types (STs) and clonal relation, respectively. The neonates in the studied cases had history of pre-maturity or low birth weight with maternal complications. All the cases were empirically treated with piperacillin–tazobactam and amikacin followed by imipenem/meropenem and vancomycin and polymyxin B as a last resort. However, all the neonates finally succumbed to the condition (100%). The studied isolates were XDR including resistance to polymyxins harboring multiple ESBL genes and carbapenemase genes (blaNDM and blaOXA−48). Hypervirulence genes were present in various combinations with rmpA/A2 genes present in all the isolates. IncFI plasmids were detected in these isolates. All belonged to ST5235. In ERIC PCR, 6 different clusters were seen. The study highlighted the emergence and burden of XDR hypervirulent isolates of K. pneumoniae causing neonatal sepsis in a tertiary care hospital.

Molecular typing of Escherichia coli strains isolated from urinary tract infection by ERIC-PCR

IntroductionEscherichia coli is a commensal-pathogenic organism with a wide range of strains. Several advanced molecular genomic-based technologies are used for detecting and identifying various strains of E. coli. Among them, the Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) technique is a fingerprint, fast and cost-effective method. The present study was aimed to determine the distribution of ERICs in E. coli strains isolated from different urine samples of patients. MethodsA total of 92 UPEC isolates were obtained from inpatients and outpatients in Besat hospital and confirmed using standard microbiological, biochemical and molecular methods. The genetic relationship of the strains was investigated using ERIC method. ResultsOverall, 27 ERIC patterns were obtained that which 14 patterns were similar between different isolates (Common type) and 13 patterns were unique (Unique type). ConclusionERIC-PCR is a simple, fast and low cost method to describe the genetic diversity of different E. coli strains including UPEC strains.

Emergence of NDM-5 Producing Carbapenem-Resistant Klebsiella aerogenes in a Pediatric Hospital in Shanghai, China.

Background: Carbapenem-resistant Klebsiella aerogenes (CRKA) has posed a serious threat for clinical anti-infective therapy. However, the molecular characteristics of CRKA in Shanghai are rarely reported.Objective: This study aimed to investigate the resistance profiles, dissemination mechanism, and molecular characteristics of CRKA strains isolated from children in a pediatric hospital, Shanghai.Method: Fifty CRKA isolates were collected in 2019. Antimicrobial susceptibility of the strains was determined by broth microdilution method. The β-lactamases and outer membrane porin genes were characterized by polymerase chain reaction (PCR). Conjugation experiments were performed to determine the transferability of the plasmids. The plasmids were typed based on their incompatibility group using the PCR-based replicon typing method. Multilocus sequence typing (MLST) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) were performed for the genetic relationship.Results: All CRKA strains showed high level of resistance to cephalosporins and carbapenems, but still susceptible to aminoglycosides, colistin, and tigecycline. Forty five of fifty isolates carried blaNDM−5 genes (45/50, 90%), alongside with other β-Lactamase genes including blaCTX−M−1, blaTEM−1, and blaSHV−11 being detected. Loss of ompK35 and ompK36 genes were observed in 14% (7/50) and 28% (14/50), respectively, with 5 isolates lacking both ompK35 and ompK36. MLST analysis demonstrated that the majority of isolates belonged to ST4 (47/50, 94%) and ERIC-PCR fingerprinting was performed to identify NDM-5-producing isolates with approximately or more than 80% similarity levels. Plasmids carrying blaNDM−5 were successfully transferred to the E. coli recipient and plasmid typing showed that IncX3 were the prevalent among CRKA isolates.Conclusions: Our finding revealed the emergence of NDM-5 producing CRKA belonging to ST4 among children in Shanghai. Further attention should be paid to control the horizontal spread of the Class B carbapenemases like NDM in children.

Occurrence and Determination of Antimicrobial Resistant Escherichia coli Isolates in Fish and Vegetables as Indicator Organism of Faecal Contamination in Dar es Salaam, Tanzania.

Escherichia coli such as E. coli O157:H7, a non-sorbitol-fermenting (NSF) E. coli, is an essential human pathogen among other common zoonotic pathogens carried by animals especially cattle. They are discharged through cattle faeces into the environment. With the increasing practice of urban farming, livestock manure is used as organic fertiliser in either fish ponds or vegetable gardens. This practice increases the risk of transmission of such pathogens to humans. This study aimed at determining the occurrence, antimicrobial resistance profiles, and genetic relatedness of E. coli isolates from manure, vegetables, and fish. Microbiological standard methods were used to isolate and identify E. coli isolates from manure, vegetable, and fish samples. Confirmed isolates on biochemical tests were tested for resistance against six antibiotics using the disc diffusion method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) typing method was used to generate fingerprints and determine the genetic relatedness of the E. coli isolates. Of 156 samples including 89 manure, 53 vegetables, and 16 fish, 36 (23.1%) samples were positive for E. coli from where a total of 48 E. coli different isolates were recovered that were subjected to antimicrobial susceptibility testing and genetic relatedness. Of these isolates, 25 (52.1%) were resistant to at least one antimicrobial agent and 12 (48.0%) showed multidrug resistance. ERIC-PCR profiles of E. coli isolates from manure, vegetables, and fish showed genetic diversity with genetic relatedness ranging from 74.5% to 100%. Nine phylogenetic clusters (I–IX) determined at 90% threshold level of genetic relatedness were identified among the isolates. This study determined the occurrence, antimicrobial resistant patterns, and genetic diversity of antimicrobial-resistant E. coli isolates from different sources. This study showed the potential of microbial health risk to humans through contamination, and hence, it is necessary to monitor and improve husbandry practices in urban farming.