Phenotypical and Genotypical Comparison of Clostridium difficile Isolated from Clinical Samples: Homebrew DNA Fingerprinting versus Antibiotic Susceptibility Testing (AST) and Clostridial Toxin Genes

Background. Clostridium (Clostridioides) difficile is recognized as the major cause of healthcare antibiotic-associated diarrhea. We surveyed a molecular epidemiological correlation between the clinical isolates from two general hospitals in Iran through clustering toxigenic types and antibiotic susceptibility testing (AST) accuracy. Methods. Study population included 460 diarrhoeic specimens from inpatients with a history of antibiotic therapy. All samples underwent enriched anaerobic culture, confirmed by detection of gluD gene with PCR. Toxin status and AST were assessed by the disk diffusion method (DDM) and minimal inhibitory concentrations (MICs) of metronidazole, vancomycin, and rifampin. C. difficile outbreak was analyzed through conventional PCR by tracing toxin genes and Homebrew pulsed-field gel electrophoresis (PFGE) for characterizing isolates within our healthcare systems. Results. A total of 29 C. difficile strains were isolated by enriched anaerobic culture from the clinical samples. Among them, 22 (4.8%) toxigenic profiles yielded toxins A and B (tcdA, tcdB) and binary toxins (cdtA, cdtB). The minimum inhibitory concentration (MIC) was 18.1% and 9% for vancomycin and metronidazole, and all isolates were susceptible to rifampicin and its minimum inhibitory concentration was at <0.003 μg/mL. The most dominant toxigenic and antibiotic-resistant “pulsotype F” was detected through PFGE combined with multiple Clostridial toxigenic pattern and AST. Conclusions. DNA fingerprinting studies represent a powerful tool in surveying hypervirulent C. difficile strains in clinical settings. Resistance to vancomycin and metronidazole, as first-line antibiotics, necessitate accomplishment of proper control strategies and also prescription of tigecycline as a more appropriate option.

Clinical Significance of Toxigenic Clostridioides difficile Growth in Stool Cultures during the Era of Nonculture Methods for the Diagnosis of C. difficile Infection

The importance of detecting toxins or toxin genes when diagnosing Clostridioides difficile infections (CDIs) or predicting the severity and outcomes of CDI has been emphasized in recent years. Although the yielding of C. difficile from stool cultures might implicate higher bacterial loads in fecal samples, in an era of nonculture methods for the standard diagnosis of CDIs, clinical significance of positive stool cultures of toxigenic C. difficile was analyzed in this study.

Molecular Epidemiology of Community-Onset Clostridioides Difficile Infections At A Tertiary Hospital In Mainland China: A Ten-Year (2010-2019) Retrospective Study

Background: Clostridioides difficile infection (CDI) is an increasingly common disease in healthcare facilities and community settings. However, there are limited reports of community-onset CDI (CO-CDI) in China. We retrospectively analyzed the molecular epidemiology of CO-CDI at a tertiary hospital over a period of 10 years. A total of 1307 stool samples from 1213 outpatients were tested by culturing. The presence of toxin genes (tcdA, tcdB, cdtA, and cdtB) were confirmed by PCR. Toxigenic strains were typed using multilocus sequence typing (MLST). Susceptibility to 9 antimicrobials was evaluated using the E-test.Results: Eighty-nine of 1213 outpatients (7.3%) had CO-CDI, 4 of these patients (4.5%) had one or more recurrence, and there were 95 strains of toxigenic C. difficile. Among these strains, 82 (86.3%) had the tcdA and tcdB genes (A+B+) and 5 of these 82 strains were positive for the binary toxin genes (cdtA and cdtB); the other 13 strains (13.7%) had the tcdB gene only (A−B+). There were 15 different STs, and the most prevalent were ST-54 (23.2%), ST-35 (16.8%), and ST-2 (13.7%). All strains were susceptible to metronidazole and vancomycin, and had low resistance to moxifloxacin and tetracycline, but had high resistance to ciprofloxacin, clindamycin, and erythromycin. Twenty-three isolates (24.2%) were multidrug-resistant.Conclusions: Outpatients with CDI were common during this period in our hospital. The C. difficile isolates had high genetic diversity. All isolates were susceptible to metronidazole and vancomycin, and nearly one quarter of all isolates had multidrug resistance.

Phenotypic and genotypic study on antibiotic resistance and pathogenic factors of Staphylococcus aureus isolates from small ruminant mastitis milk in South of Italy (Sicily)

Staphyloccoccus aureus is the major cause of mastitis in small ruminants in the Mediterranean farms causing severe losses to dairy industry. Antibiotic treatment has been the most common approach to control these infections. Aim of this study was to investigate antimicrobial resistance (AMR), virulence factors and biofilm-related genes of 84 Sicilian strains of S. aureus isolated from sheep and goats milk during two different periods δT1 (2006-2009) and δT2 (2013-2015). Kirby Bauer method and Polymerase Chain Reaction (PCR) were utilized to monitor AMR and related genes (mecA, tetK, tetM, ermA, ermC). Moreover, toxin genes (tsst-1, sea-see, seg-sej, and sep) and biofilm genes (bap, ica, sasC) were studied. Twenty-six isolates (30.9%) showed multidrug resistance. The two groups showed similar results with exception for higher values of resistance for tilmicosin and lower for sulfamethoxazole and vancomycin of the second group. MecA gene was detected in one isolate. Tetracycline resistance was higher than 20%, with an increase in δT2 group. Toxin genes were found in 5 isolates (5.9%), belonging of δT2 group, while 57 of isolates (67.8%) showed biofilm related genes. The high presence of multi-resistant isolates suggests the need of more responsible use of antibiotic therapy for the control of these infections.

The Possible Transmission and Potential Enterotoxicity of Bacillus cereus on Lettuce Farms in Five Chinese Provinces

Bacillus cereus is a well-characterized human pathogen that produces toxins associated with diarrheal and emetic foodborne diseases. To investigate the possible transmission of B. cereus on lettuce farms in China and determine its enterotoxicity, (I) a total of 524 samples (lettuce: 332, soil: 69, water: 57, manure: 57, pesticide: 9) were collected from 46 lettuce farms in five Chinese provinces, (II) multilocus sequence typing (MLST) was used to classify B. cereus isolates and for trace analysis, and (III) the presence of toxin genes and enterotoxins (Hbl and Nhe) was detected in 68 strains. The results showed that one hundred and sixty-one lettuce samples (48.5%) tested positive for B. cereus at levels ranging from 10 to 5.3 × 104 CFU/g. Among the environmental sample categories surveyed, the highest positive rate was that of the pesticide samples at 55.6%, followed by soil samples at 52.2% and manure samples at 12.3%. Moreover, one hundred isolates of B. cereus yielded 68 different sequence types (STs) and were classified into five phylogenetic clades. Furthermore, Nhe toxin genes (nheA, nheB, nheC) were broadly distributed and identified in all 68 strains (100%), while Hbl toxin genes (hblA, hblC, hblD) were present in 61 strains (89.7%), entFM was detected in 62 strains (91.2%), and cytK was found in 29 strains (42.6%). All strains were negative for ces. As for the enterotoxin, Nhe was observed in all 68 isolates carrying nheB, while Hbl was present in 76.5% (52/68) of the strains harboring hblC. This study is the first report of possible B. cereus transmission and of its potential enterotoxicity on lettuce farms in China. The results showed that soil and pesticides are the main sources of B. cereus on lettuce farms in China, and the possible transmission routes are as follows: soil-lettuce, manure-lettuce, pesticide-lettuce, manure-soil-lettuce, and water-manure-soil-lettuce. Furthermore, the B. cereus isolates, whether from lettuce or the environment, pose a potential risk to health.

Prevalence, Virulence, and Antimicrobial Resistance of Campylobacter Species Isolated From Carcasses of Camels Slaughtered in Slaughterhouses of Chaharmahal and Bakhtiari Province, 2018-2019

Background and aims: Gastritis is basically caused by Campylobacter coli and jejuni, and usually occures after the consumption of raw animal products. Methods: This study investigated the prevalence, virulence, and antimicrobial resistance of Campylobacter species isolated from slaughtered animals in Juneqan, Farrokhshahr, Saman, and Lordegan slaughterhouses in Chaharmahal and Bakhtiari Province of Iran. From 40 camels, 5 samples of liver, neck meat, kidney, heart, and rectal contents were taken from each carcass. The obtained samples were cultured and then the PCR was performed for them and, finally, the toxin genes of virulence and resistance against antibiotics were examined. Results: Out of 19 Campylobacter specimens isolated, 8 specimens were coli and 11 ones were jejuni. It was also found that the infection with Campylobacter in the carcasses was the highest in warmer seasons. Conclusion: The carcasses of slaughtered animals in slaughterhouses were likely a potential reservoir for coli and jejuni species, and their viscera and meat could have transmitted these bacteria to humans and animals.

Molecular epidemiology of community-onset Clostridioides difficile infections at a tertiary hospital in mainland China: A ten-year (2010-2019) retrospective study

Background Clostridioides difficile infection (CDI) is an increasingly common disease in healthcare facilities and community settings. However, there are limited reports of community-onset CDI (CO-CDI) in China. We retrospectively analyzed the molecular epidemiology of CO-CDI at a tertiary hospital over a period of 10 years. Methods A total of 1307 stool samples from 1213 outpatients were tested by culturing. The presence of toxin genes (tcd A, tcd B, cdtA and cdtB) were confirmed by PCR. Toxigenic strains were typed using multilocus sequence typing (MLST). Susceptibility to 9 antimicrobials was evaluated using the E-test. Results Eighty-nine of 1213 outpatients (7.3%) had CO-CDI, 4 of these patients (4.5%) had one or more recurrence, and there were 95 strains of toxigenic C. difficile. Among these strains, 82 (86.3%) had the tcdA and tcdB genes (A + B+) and 5 of these 82 strains were positive for the binary toxin genes (cdtA and cdtB); the other 13 strains (13.7%) had the tcdB gene only (A-B+). There were 15 different STs and the most prevalent were ST-54 (23.2%), ST-35 (16.8%), and ST-2 (13.7%). All strains were susceptible to metronidazole and vancomycin, and had low resistance to moxifloxacin and tetracycline, but had high resistance to ciprofloxacin, clindamycin, and erythromycin. Twenty-three isolates (24.2%) were multidrug-resistant. Conclusions Outpatients with CDI were common during this period in our hospital. The C. difficile isolates had high genetic diversity. All isolates were susceptible to metronidazole and vancomycin, and nearly one quarter of all isolates had multidrug resistance.