Phenotypic and genotypic analysis of antimicrobial resistance in Nocardia species.

BACKGROUND: Antimicrobial resistance is common in Nocardia species but data regarding the molecular mechanisms beyond their resistance traits are limited. Our study aimed to determine the species distribution, the antimicrobial susceptibility profiles, and investigate the associations between the resistance traits and their genotypic determinants. METHODS: The study included 138 clinical strains of Nocardia from nine Israeli microbiology laboratories. MIC values of 12 antimicrobial agents were determined using broth microdilution. WGS was performed on 129 isolates of the eight predominant species. Bioinformatic analysis included phylogeny and determination of antimicrobial resistance genes and mutations. RESULTS: Among the isolates, Nocardia cyriacigeorgica was the most common species (36%), followed by Nocardia farcinica (16%), Nocardia wallacei (13%), Nocardia abscessus (9%) and Nocardia brasiliensis (8%). Linezolid was active against all isolates, followed by trimethoprim/sulfamethoxazole (93%) and amikacin (91%). Resistance to other antibiotics was species-specific, often associated with the presence of resistance genes or mutations: (1) aph(2″) in N. farcinica and N. wallacei (resistance to tobramycin); (ii) blaAST-1 in N. cyriacigeorgica and Nocardia neocaledoniensis (resistance to amoxicillin/clavulanate); (iii) blaFAR-1 in N. farcinica (resistance to ceftriaxone); (iv) Ser83Ala substitution in the gyrA gene in four species (resistance to ciprofloxacin); and (v) the 16S rRNA m1A1408 methyltransferase in N. wallacei isolates (correlating with amikacin resistance). CONCLUSIONS: Our study provides a comprehensive understanding of Nocardia species diversity, antibiotic resistance patterns, and the molecular basis of antimicrobial resistance. Resistance appears to follow species-related patterns, suggesting a lesser role for de novo evolution or transmission of antimicrobial resistance.

Trends in the Epidemiology of Campylobacteriosis in Israel (1999-2012).

The objective of this study was to examine the recent trends in the epidemiology of campylobacteriosis in Israel. A Sentinel Laboratory-Based Surveillance Network for Bacterial Enteric Diseases was established in Israel by the Israel Center for Disease Control (ICDC). This network generated data on subjects from whom Campylobacter spp. was isolated in community and hospital laboratories. Further characterization of the isolates was done at the Campylobacter National Reference Laboratory. Data from these two sources were integrated and analyzed at the ICDC. Between 1999 and 2012, 40,978 Campylobacter stool isolates were reported to the ICDC by the sentinel laboratories. The incidence rate of campylobacteriosis increased from 65.7 per 100,000 in 1999 to 101.7 per 100,000 in 2012. This increase resulted from a significant rise in the incidence of campylobacteriosis in the Jewish population which, since 2009, surpassed the consistent higher incidence of the disease in Israeli Arabs. The peak morbidity in Israel consistently occurred in late spring, with a risk excess in males compared with females, in younger age groups and earlier in the life span among Arabs than among Jews and others. These results suggest that further analytical studies should be carried out to identify risk factors responsible for the increased incidence of campylobacteriosis and better direct prevention and control of the disease in Israel.

Characterization of community-acquired Clostridioides difficile strains in Israel, 2020-2022.

Background: The prevalence of community-acquired Clostridioides difficile infection (CA-CDI) has been rising, due to changes in antibiotics prescribing practices, emergence of hypervirulent strains and improved diagnostics. This study explored CA-CDI epidemiology by examining strain diversity and virulence factors of CA-CDI isolates collected across several geographical regions in Israel. Methods: Stool samples of 126 CA-CDI patients were subjected to PCR and an immunoassay to identify toxin genes and proteins, respectively. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Biofilm production was assessed by crystal violet-based assay. Minimum inhibitory concentration was determined using the Etest technique or agar dilution. WGS and multi-locus sequence typing (MLST) were used to classify strains and investigate genetic diversity. Results: Sequence types (ST) 2 (17, 13.5%), ST42 (13, 10.3%), ST104 (10, 8%) and ST11 (9, 7.1%) were the most common. All (117, 92.8%) but ST11 belonged to Clade 1. No associations were found between ST and gender, geographic area or antibiotic susceptibility. Although all strains harbored toxins genes, 34 (27%) produced toxin A only, and 54 (42.9%) strains produced toxin B only; 38 (30.2%) produced both toxins. Most isolates were biofilm-producers (118, 93.6%), primarily weak producers (83/118, 70.3%). ST was significantly associated with both biofilm and toxin production. Conclusion: C. difficile isolates in Israel community exhibit high ST diversity, with no dominant strain. Other factors may influence the clinical outcomes of CDI such as toxin production, antibiotic resistance and biofilm production. Further studies are needed to better understand the dynamics and influence of these factors on CA-CDI.

Trends in the Epidemiology of Non-Typhoidal Salmonellosis in Israel between 2010 and 2021.

Non-typhoidal salmonellosis (NTS) is one of the most common foodborne diseases worldwide. In this study, we aimed to analyze trends in the epidemiology of NTS in the last decade in Israel. Laboratory-confirmed cases of NTS at eight sentinel laboratories were reported to the Israel Sentinel Laboratory-Based Surveillance Network, integrated with the serotype identification performed at the Salmonella National Reference Laboratory of the Ministry of Health. The decrease in NTS incidence since 1999 continued between 2010 and 2014 (16.1 per 100,000 in 2014) and was interrupted by a rise between 2015 and 2017 (39.1 per 100,000 in 2017) associated with outbreaks of Salmonella Enteritidis. The incidence of NTS dropped again thereafter (21.4 per 100,000 in 2021). The 0-4 age group was the most affected by NTS (55.5% of the cases) throughout the surveillance period. The age-adjusted incidence rates were consistently high in the summer months (June-September) and low in the winter months (December-February). The overall decrease in the incidence of NTS in Israel since 1999 was temporarily interrupted in the last decade by country-wide outbreaks involving emerging or re-emerging Salmonella serotypes. Control measures should be enhanced for all risk points of food chain transmission of Salmonella spp. to further reduce the NTS morbidity in Israel.

Tsukamurella pulmonis conjunctivitis in patients with an underlying nasolacrimal duct obstruction - report of two cases.

Tsukamurella pulmonis ( Actinobacteria ), a Gram-positive, obligate aerobic and weakly or variably acid-fast bacterium, is an opportunistic pathogen. Here we report two cases of conjunctivitis caused by T. pulmonis . Both patients had a previous history of nasolacrimal duct obstruction (NLDO). Isolation of T. pulmonis was performed on chocolate, tryptic soy blood and Columbia nalidixic agars. After 24 h of incubation, odourless, white-greyish, membrane-like colonies were observed. The VITEK-2 bacterial identifier system failed to identify the species, while Vitek-MS matrix-assisted laser desorption ionization time-of-flight technology, successfully identified the isolate from case 2 but not from case 1. Final identification was verified using 16S rRNA gene sequencing. An antibiogram was performed and according to the results cefazoline in addition to vancomycin eye drops for 5 days, were suggested as a treatment in case 1. In case 2 the infection was ended without treatment. This is the first report of Tsukamurella as a pathogen that causes conjunctivitis in patients with NLDO.

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals.

The BNT162b2 mRNA vaccine is highly effective against SARS-CoV-2. However, apprehension exists that variants of concern (VOCs) may evade vaccine protection, due to evidence of reduced neutralization of the VOCs B.1.1.7 and B.1.351 by vaccine sera in laboratory assays. We performed a matched cohort study to examine the distribution of VOCs in infections of BNT162b2 mRNA vaccinees from Clalit Health Services (Israel) using viral genomic sequencing, and hypothesized that if vaccine effectiveness against a VOC is reduced, its proportion among breakthrough cases would be higher than in unvaccinated controls. Analyzing 813 viral genome sequences from nasopharyngeal swabs, we showed that vaccinees who tested positive at least 7 days after the second dose were disproportionally infected with B.1.351, compared with controls. Those who tested positive between 2 weeks after the first dose and 6 days after the second dose were disproportionally infected by B.1.1.7. These findings suggest reduced vaccine effectiveness against both VOCs within particular time windows. Our results emphasize the importance of rigorously tracking viral variants, and of increasing vaccination to prevent the spread of VOCs.

Clinical characteristics and outcomes of patients with Escherichia coli in airway samples.

PURPOSES: Escherichia coli is one of the most common pathogens in nosocomial and community-acquired infections, but is an uncommon respiratory pathogen. However, this pathogen may at times be seen in respiratory secretions. The study aims to determine the clinical and prognostic value of E. coli in respiratory secretions. METHODS: Cultures of respiratory secretions from hospitalized and outpatients between 2009 and 2016 were screened for isolation of E. coli. We defined three groups of patients: "Sensitive (S)"-growth of E. coli sensitive to all antimicrobials tested; Intermediate (I)-resistant to 1-2 antimicrobial classes; and "Resistant (R)"-resistant to at least three antibiotic classes. We compared factors associated with resistant strains and outcomes between the groups. RESULTS: Eighty patients with E. coli isolates from respiratory secretions were identified while screening 177 712 (4.5: 10 000 samples). Of the E. Coli-positive cultures, 11 were from ambulatory patients, 31 patients were hospitalized and 37 were hospitalized and intubated. Ten people had bronchiectasis and 29 had COPD. Patients with resistant E. coli had significantly more hospitalization days prior to positive culture (S = 1.2 ± 1.89 days, I = 1.23 ± 1.5 days and R = 3.7 ± 5.4 days, respectively; P = 0.002). Mortality was higher in patients with a resistant strain (R) versus (I) or (S) (76.7%, 31.8% and 26.7%, respectively; P < 0.0001) and remained significantly elevated after correction for prior hospital days. CONCLUSIONS: Pulmonary infection due to E. coli is uncommon. Isolation of resistant E. coli is associated with length of previous hospitalization, elevated mortality and may be viewed as a nosocomial pathogen.

Molecular typing of Cryptosporidium in Israel.

Cryptosporidium is a protozoan parasite associated with gastrointestinal illness. In immune-compromised individuals, the infection may become life-threatening. Cryptosporidiosis is a mandatory-reported disease but little was known about its prevalence and associated morbidity in Israel. Currently, laboratory diagnosis is based on microscopy or copro-antigen tests and the disease is underreported. Molecular assays, which are more sensitive and specific, are now increasingly used for identification and screening. Here, the molecular epidemiology of cryptosporidiosis is explored for the first time. Samples from 33 patients infected during an outbreak of 146 laboratory confirmed cases that occurred in Haifa and Western Galilee in 2015 were genotyped, as well as samples from 36 patients sporadically infected during 2014-2018 in different regions. The results suggest that Cryptosporidium subtypes found in Israel are more similar to those reported in the neighboring countries Jordan and Egypt than in European countries. C. hominis was the predominant species in the center and the north of Israel, implicating human-to-human transmission. C. hominis IeA11G3T3 was the most prevalent subtype contributing to morbidity.

Otitis Media Caused by V. cholerae O100: A Case Report and Review of the Literature.

Infections due to Vibrio cholerae are rarely documented in Israel. Here we report a case of recurrent otitis media in a young male, caused by V. cholerae non-O1/O139. This extra-intestinal infection was caused by V. cholerae O100 and has been associated with freshwater exposure and travel. Symptoms of chronic periodic earaches along with purulent exudate began about one week after the patient suffered a water skiing accident on a river in Australia. The condition lasted for three years, until his ear exudate was examined in a clinical laboratory, diagnosed and treated. Five bacterial isolates were identified as V. cholerae O100. The isolates were screened for genetic characteristics and were found positive for the presence of hapA, hlyA, and ompU virulence genes. All isolates were negative for the presence of ctxA. Based on antibiogram susceptibility testing, ciprofloxacin ear drops were used until the patient's symptoms disappeared. This case demonstrates that exposure to freshwater can cause otitis media by V. cholerae non-O1/O139 in young and otherwise healthy humans.

Evaluation of the NanoCHIP® Gastrointestinal Panel (GIP) Test for Simultaneous Detection of Parasitic and Bacterial Enteric Pathogens in Fecal Specimens.

Infectious gastroenteritis is a global health problem associated with high morbidity and mortality rates. Rapid and accurate diagnosis is crucial to allow appropriate and timely treatment. Current laboratory stool testing has a long turnaround time (TAT) and demands highly qualified personnel and multiple techniques. The need for high throughput and the number of possible enteric pathogens compels the implementation of a molecular approach which uses multiplex technology, without compromising performance requirements. In this work we evaluated the feasibility of the NanoCHIP® Gastrointestinal Panel (GIP) (Savyon Diagnostics, Ashdod, IL), a molecular microarray-based screening test, to be used in the routine workflow of our laboratory, a big outpatient microbiology laboratory. The NanoCHIP® GIP test provides simultaneous detection of nine major enteric bacteria and parasites: Campylobacter spp., Salmonella spp., Shigella spp., Giardia sp., Cryptosporidium spp., Entamoeba histolytica, Entamoeba dispar, Dientamoeba fragilis, and Blastocystis spp. The required high-throughput was obtained by the NanoCHIP® detection system together with the MagNA Pure 96 DNA purification system (Roche Diagnostics Ltd., Switzerland). This combined system has demonstrated a higher sensitivity and detection yield compared to the conventional methods in both, retrospective and prospective samples. The identification of multiple parasites and bacteria in a single test also enabled increased efficiency of detecting mixed infections, as well as reduced hands-on time and work load. In conclusion, the combination of these two automated systems is a proper response to the laboratory needs in terms of improving laboratory workflow, turn-around-time, minimizing human errors and can be efficiently integrated in the routine work of the laboratory.

Rapid diagnosis of acanthamoeba keratitis using non-nutrient agar with a lawn of E. coli.

BACKGROUND: A patient presented with a corneal foreign body in his only eye. He was treated with prophylactic antibiotics and sent home, but deteriorated. FINDINGS: He returned to the hospital 5 days later, and on slit-lamp examination, there was ciliary injection, corneal oedema and a 1 mm × 1 mm corneal abscess with mild anterior uveitis. Corneal scrapings were taken for culture on a non-nutrient agar with a lawn of Escherichia coli, on chocolate agar and on blood agar. He was treated with fortified gentamicin and cefazolin drops. He improved and was discharged 4 days after admission. On day 5, the culture results showed acanthamoeba. He was brought back to the hospital and treated with hourly chlorhexidine drops, ofloxacin six times daily and neomycin/dexamethasone drops once daily. On day 7, he was discharged to continue treatment at home, at which time his visual acuity in that eye was 6/9, and slit-lamp examination showed punctate keratitis and a stromal opacity with mild peripheral infiltration. CONCLUSIONS: Culture on non-nutrient agar with a lawn of E. coli is a rapid, reliable and less invasive alternative to corneal biopsy for the diagnosis of acanthamoeba infection. We suggest using this method where acanthamoeba is suspected. Owing to the risk of corneal abscess, orthokeratology should be avoided in an amblyopic patient or an only eye. Acanthamoeba infection may be masked by other eye diseases.

A molecular study on the prevalence and virulence potential of Aeromonas spp. recovered from patients suffering from diarrhea in Israel.

BACKGROUND: Species of the genus Aeromonas are native inhabitants of aquatic environments and have recently been considered emerging human pathogens. Although the gastrointestinal tract is by far the most common anatomic site from which aeromonads are recovered, their role as etiologic agents of bacterial diarrhea is still disputed. Aeromonas-associated diarrhea is a phenomenon occurring worldwide; however, the exact prevalence of Aeromonas infections on a global scale is unknown. METHODOLOGY/PRINCIPAL FINDINGS: The prevalence and virulence potential of Aeromonas in patients suffering from diarrhea in Israel was studied using molecular methods. 1,033 diarrheal stools were sampled between April and September 2010 and Aeromonas species were identified in 17 (∼2%) patients by sequencing the rpoD gene. Aeromonas species identity and abundance was: A. caviae (65%), A. veronii (29%) and Aeromonas taiwanensis (6%). This is the first clinical record of A. taiwanensis as a diarrheal causative since its recent discovery from a wound infection in a patient in Taiwan. Most of the patients (77%) from which Aeromonas species were isolated were negative for any other pathogens. The patients ranged from 1 to 92 years in age. Aeromonas isolates were found to possess different virulence-associated genes: ahpB (88%), pla/lip/lipH3/apl-1 (71%), act/hlyA/aerA (35%), alt (18%), ast (6%), fla (65%), lafA (41%), TTSS ascV (12%), TTSS ascF-ascG (12%), TTSS-dependent ADP-ribosylating toxins aexU (41%) and aexT (6%) in various combinations. Most of the identified strains were resistant to beta-lactam antibiotics but susceptible to third-generation cephalosporin antibiotics. CONCLUSIONS: Aeromonas may be a causative agent of diarrhea in patients in Israel and therefore should be included in routine bacteriological screenings.

Identification of the emerging pathogen Vibrio vulnificus biotype 3 by commercially available phenotypic methods.

Identification of the emerging pathogen Vibrio vulnificus biotype 3 has become a challenge for clinical laboratories in the last few years. In this study, the abilities of five commercial systems to identify this new species have been evaluated for the first time, using a unique collection of strains. Fifty-one well-documented wild strains of V. vulnificus biotype 3 were processed using API 20 NE, GNI+ Vitek 1 cards, ID-GNB Vitek 2 cards, Neg Combo 20 Microscan panels, and NMIC/ID-5 BD Phoenix panels. The numbers of strains identified as V. vulnificus by ID-GNB, NMIC/ID-5, and GNI+ were 50 (98.0%), 46 (90.2%), and 7 (13.7%), respectively. Neg Combo 20 Microscan panels and API 20 NE were unable to identify any of the strains of this emerging pathogen to the species level and mostly misidentifies them as other species of the Vibrionaceae family. Data on the phenotypic pattern of V. vulnificus biotype 3 when processed in all five systems as presented here could help clinical laboratories in identifying this new pathogen.