Enterobacterial Repetitive Intergenic Consensus Polymerase Research Articles

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran

ObjectivesIn the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis.ResultsIn total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX−M−15 gene (66.1%) followed by blaCTX−M (45.8%), blaCTX−M−14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Microbiological quality of irrigation water on highly diverse fresh produce smallholder farms: elucidating environmental routes of contamination.

To evaluate the microbiological safety, potential multidrug-resistant bacterial presence and genetic relatedness (DNA fingerprints) of Escherichia coli isolated from the water-soil-plant nexus on highly diverse fresh produce smallholder farms. Irrigation water (n=44), soil (n=85), and fresh produce (n=95) samples from six smallholder farms with different production systems were analysed for hygiene indicator bacterial counts and the presence of shigatoxigenic E. coli and Salmonella spp. using standard microbiological methods. Identities of isolates were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the genetic relatedness of the E. coli isolates determined using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) analysis. Irrigation water E. coli levels ranged between 0 and 3.45 log MPN/100 ml-1 with five farms having acceptable levels according to the World Health Organization limit (3 log MPN/100 ml-1). Fresh produce samples on four farms (n=65) harboured E. coli at low levels (<1 log CFU/g-1) except for one sample from kale, spring onion, green pepper, onion, and two tomato samples, which exceeded international acceptable limits (100 CFU/g-1). Only one baby carrot fresh produce sample tested positive for Salmonella spp. Of the 224 samples, E. coli isolates were identified in 40% (n=90) of all water, soil, and fresh produce types after enrichment. Additionally, the DNA fingerprints of E. coli isolates from the water-soil-plant nexus of each respective farm clustered together at high similarity values (>90%), with all phenotypically characterized as multidrug-resistant. The clustering of E. coli isolated throughout the water-soil-plant nexus, implicated irrigation water in fresh produce contamination. Highlighting the importance of complying with irrigation water microbiological quality guidelines to limit the spread of potential foodborne pathogens throughout the fresh produce supply chain.

Molecular typing of clinical multidrug-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is an opportunistic pathogen which is an important cause of hospital-acquired and antibiotic resistance infections. Therefore, this study aimed to determine the frequency of resistance to antibiotics, as well as the molecular typing of the associated isolates, and compare multiple-locus VNTR analysis (MLVA) and Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) methods to specify the degree to which distinctions can be separated from each other. One hundred K. pneumoniae isolates were obtained from different sources of infections from patients admitted to hospitals. Antibiotic susceptibility testing was then performed by applying the Kirby-Bauer disk diffusion method. Typing of K. pneumoniae was done by utilizing MLVA and ERIC-PCR methods. Eighty-six multidrug-resistant (MDR) K. pneumoniae isolates were identified, which resistance to ampicillin, trimethoprim/sulfamethoxazole, and ceftriaxone was the most frequent in the considered isolates (100, 93, and 93%, respectively). A total of 50 different antibiotic susceptibility patterns were observed among the MDR K. pneumonia, with the most frequent pattern being resistance to all antibiotics (12.79%) and resistance to all antibiotics except amikacin (10.47%). The isolates were then divided into 37 different MLVA types and seven clonal complexes were obtained from the minimum spanning tree analysis. Finally, the isolates were assigned to 38 different ERIC types. The discriminatory power of MLVA and ERIC methods also showed a value of 0.958, and 0.974. Both PCR-typing methods with phenotypic patterns can be useful for the epidemiological typing of K. pneumoniae isolates with the highest performance in discriminating isolates.

Serotype and genotyping of &lt;i&gt;Salmonella&lt;/i&gt; sp. isolated from street foods in N’Djaména, Chad

Group foodborne illness infections with Salmonella are often caused by the simultaneous consumption by several people of heavily contaminated food. The objective of this work was to determine the phylogenetic relationships between dietary strains of Salmonella sp. isolated from our samples and confirmed clinical strains. A total of 447 samples were collected and analyzed using standard food microbiology methods. Enterobacterial Repetitive Intergenic Consensus-Polymerase chain Reaction (ERIC-PCR) technique allowed confirmation that all our strains belong to the genus Salmonella. From ten (10) strains characterized, eight (8) different genotype profiles were identified. The different DNA fragments of the isolates were generated by ERIC-PCR and then revealed on the agarose gel and had bands ranging from 2 to 6 bands with sizes between 150 and 1500 bp. The two food strains of S. Mbadaka (1 and 2) presented identical profiles. However, it is noted that two (2) strains of S. Anatum had different ERIC-PCR profiles. There is also a very high resemblance between the strain of S. Anatum (strain 5) and the confirmed clinical strains (H5: Salmonella Typhi). The presence of risky germs in the food analyzed poses a public health problem and should call on the competent authorities and prompt the taking of corrective measures.

Salmonella enterica serovars linked with poultry in India: antibiotic resistance profiles and carriage of virulence genes.

Salmonella is an important poultry pathogen with zoonotic potential. Being a foodborne pathogen, Salmonella-contaminated poultry products can act as the major source of infection in humans. In India, limited studies have addressed the diversity of Salmonella strains of poultry origin. This study represented 26 strains belonging to Salmonella serovars Typhimurium, Infantis, Virchow, Kentucky, and Agona. The strains were tested for resistance to 14 different antimicrobial agents using the Kirby-Bauer disk-diffusion assay. The presence of the invA, hilA, agfA, lpfA, sopE, and spvC virulence genes was assessed by polymerase chain reaction (PCR), and the genetic diversity was assessed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR). The highest resistance to tetracycline (n = 17; 65.38%) followed by nalidixic acid (n = 16; 61.53%) was detected among the strains. Among the strains (n = 17) phenotypically resistant to tetracycline, 94% (n = 16) were also positive for the tetA gene. Based on the presence of virulence genes, the strains were characterized into three virulence profiles (PI, P2, and P3). Among the investigated virulence genes, invA, hilA, agfA, and lpfA were present inall strains. The sopE gene was mostly associated with serovars Virchow (n = 3; 100%) and Typhimurium (n = 8; 80%), whereas spvC gene was exclusive for two Typhimurium strains that lacked sopE gene. ERIC-PCR profiling indicated clusters correlating their serovar, geographical, and farm origins. These results demonstrate that Salmonella isolates with a wide genetic range, antibiotic resistance, and virulence characteristics can colonize poultry. The presence of such strains is crucial for both food safety and public health.

Clinical and epidemiological characteristics of multi-drug resistant Enterobacterales isolated from King Fahad Hospital of the University, AlKhobar, Saudi Arabia.

Multi-drug resistant (MDR) Enterobacterales remain a major clinical problem. Infections caused by carbapenem-resistant strains are particularly difficult to treat. This study aimed to assess the clinical and epidemiological characteristics of MDR Enterobacterales isolates. A total of 154 non-repetitive clinical isolates, including Escherichia coli (n = 66), Klebsiella pneumoniae (n = 70), and other Enterobacterales (n = 18), were collected from the Diagnostic Microbiology Laboratory at King Fahad Hospital of the University. Most E. coli isolates were collected from urine specimens (n = 50, 75.8%) and resistance against the third and fourth-generation cephalosporins (ceftriaxone, ceftazidime, cefixime, and cefepime) and fluoroquinolones (ciprofloxacin and levofloxacin) was assessed. Clonal relatedness analysis using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) revealed two clones (E. coli A and B), each comprising two strains. Most K. pneumoniae samples were collected from respiratory specimens (27.1%, 20 samples), and the strains showed overall resistance to most of the antimicrobials tested (54%‒100%). Moreover, clonal-relatedness analysis using ERIC-PCR revealed seven major clones of K. pneumoniae. These findings suggest nosocomial transmission among some identical strains and emphasize the importance of strict compliance with infection prevention and control policies and regulations. Environmental reservoirs could facilitate this indirect transmission, which needs to be investigated.

Isolation and Characterization of Cefotaxime and Ciprofloxacin Co-Resistant Escherichia coli in Retail Chicken Carcasses

Transmission of antimicrobial-resistant bacteria to humans through the food chain is of great importance for public health. In this study, it was aimed to isolate and characterize the cefotaxime and ciprofloxacin-resistant Escherichia coli in retail chicken meat samples sold in Hatay. The isolates were subjected to phylogenetic group typing and antimicrobial susceptibility testing. The genetic relatedness of the isolates was determined using Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) technique. The isolates were also screened for the presence of both antimicrobial and plasmid-mediated quinolone resistance (PMQR) genes by PCR. Cefotaxime and ciprofloxacin co-resistant E. coli isolates with diverse genetic origins were recovered in 42.3% (22/52) of retail chicken carcasses. The E. coli isolates belonged to the phylogenetic group D2 were dominant (40.9%, 9/22), followed by B1 (27.3%, 6/22), B23 (18.2%, 4/22), and A1 (13.6%, 3/22), respectively. Based on dendrogram analysis, the ERIC-PCR method differentiated the isolates into 10 clusters (I-X). The multidrug resistance (MDR) was observed in 81.8% (18/22) of the isolates. PMQR determinants were not identified in any isolates tested. Molecular analysis revealed one or more β-lactamase-encoding genes in all isolates as a single or in combination: blaCTX-M-blaTEM (n=5), blaCMY-2 (n=5), blaCTX-M (n=5), blaCMY-2-blaSHV (n=3), blaCMY-2-blaTEM (n=3), and blaCTX-M-blaCMY-2 (n=1). This study highlights that retail chicken meat is an important reservoir of cefotaxime and ciprofloxacin co-resistant E. coli isolates. It is necessary to evaluate their contribution to the community and hospital infections.

Ecological incidence, genetic diversity, multidrug resistance of Salmonella enteritidis recovered from broiler and layer chicken farms

Salmonella is a significant foodborne pathogen that has a significant impact on public health, and different strains of multidrug resistance (MDR) have been identified in this genus. This study used a combination of phenotypic and genotypic approaches to identify distinct Salmonella species collected from poultry broiler and layer farms, and antibiotic sensitivity testing was performed on these species. A total of 56 Salmonella isolates were serotyped, and phenotypic antibiotic resistance was determined for each strain. The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) method was also used to provide a genotypic description, from which a dendrogram was constructed and the most likely phylogenetic relationships were applied. Salmonella isolates were detected in 20 (17%) out of 117 samples collected from small-scale broiler flocks. Salmonella isolates were classified as MDR strains after showing tolerance to four antibiotics, but no resistance to cloxacillin, streptomycin, vancomycin, or netilmicin was observed. From a genotypic perspective, these strains lack dfrD, parC, and blasfo-1 resistant genes, while harboring blactx-M, blaDHA-L, qnrA, qnrB, qnrS, gyrA, ermA, ermB, ermC, ermTR, mefA, msrA, tet A, tet B, tet L, tet M resistance genes. The genotyping results obtained with ERIC-PCR allowed isolates to be classified based on the source of recovery. It was determined that Salmonella strains displayed MDR, and many genes associated with them. Additionally, the ERIC-PCR procedure aided in the generation of clusters with biological significance. Extensive research on Salmonella serotypes is warranted, along with the implementation of long-term surveillance programs to monitor MDR Salmonella serotypes in avian-derived foods.

Endogenous Bacteremia Caused by Intestinal Colonization of Carbapenem-Resistant Enterobacteriaceae (CRE) in Immunocompromised Children.

Carbapenem-resistant Enterobacteriaceae (CRE) infection is life-threatening, especially for immunocompromised children. The source tracking of CRE could prevent bacteremia during hospitalization. In this study, the intestinal colonization of CRE and their translocation to blood were investigated. Stool samples from immunocompromised pediatric patients were collected after admission, and secondary stool and blood samples were collected in case of fever. After CRE phonotypic detection, the OXA-48, NDM-1, VIM, IMP, and KPC genes were detected by PCR. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) was used to determine the phylogenic relatedness of the blood and fecal isolates. Bacteremia was recorded in 71.4% of the patients. Enterobacteriaceae spp. were recorded in 100% of the stool samples and 31% of the blood samples. The correlation between the length of stay (LOS), days of fever, chemotherapy regimens, and death rate was significant (p-value ≤ 0.05). OXA-48 was present in all CRE isolates in both the primary and the secondary stool samples and the blood samples. According to the phylogenetic data, 58.33% of the patients with bacteremia had identical blood and stool isolates. The death rate was 24.4% in children with CRE bacteremia. The primary intestinal colonization with CRE in immunocompromised pediatrics and their translocation to blood was established in this study. The implementation of infection control programs and the application of infection prevention strategies for immunocompromised children is necessary.

Microbiological Characterization and Clinical Facets of Elizabethkingia Bloodstream Infections in a Tertiary Care Hospital of Eastern India.

Elizabethkingia is an emerging non-fermenting Gram-negative bacillus (NFGNB) causing bloodstream infections (BSI) associated with high mortality. It demonstrates a unique antimicrobial profile in showing susceptibility to antimicrobials effective against Gram-positive bacteria. This study was undertaken to determine the overall frequency of Elizabethkingia BSI, associated risk factors, microbiological susceptibility, and clonal relationship of Elizabethkingia isolates using Enterobacterial Repetitive Intergenic ConsensusPolymerase Chain Reaction (ERIC-PCR). Elizabethkingia isolates obtained from the blood culture of admitted patients (August 2020-December 2021) were identified by the VITEK 2 system and subjected to an antimicrobial susceptibility test by standard procedures. Demographics, co-morbidities, risk factors for survival, and outcome were summarized and analyzed by Chi-square test, Kaplan-Meier curve, and Cox regression. Clonal relatedness between Elizabethkingia isolates was analyzed using ERIC‑PCR fingerprinting with the "PAST: Paleontological statistics software package". Of 13,747 blood samples received during the study period, 13.59% were culture positive, and 14.60% were NFGNBs. The frequency of Elizabethkingia spp. among all NFGNBs in BSI was 29.30%, and the overall prevalence in BSI was 4.21%. In patients with Elizabethkingia BSI, Foley's catheter was present in 81.25% of the cases. 100% susceptibility was observed to linezolid, followed by vancomycin (98.75%) and chloramphenicol (89.5%). The 30-day mortality rate in the patients of Elizabethkingia BSI was 26.25%. The Presence of COVID-19, pneumonia, diabetes mellitus (DM), mechanical ventilation (MV), and prior antibiotics were significantly different (p<0.05) between the survival and death groups. ERIC-PCR profile dendrogram of Elizabethkingia isolates showed ten major clusters indicating high genetic diversity. Elizabethkingia was responsible for one-third of NFGNB BSI in a single-center study, with approximately 26% of 30-day all-cause mortality. Most isolates were susceptible to linezolid, vancomycin, and chloramphenicol. COVID-19 was the most significant risk factor associated with mortality. ERIC-PCR of Elizabethkingia isolates exhibited high genetic diversity.

Isolation and Molecular Characterization of Corynebacterium pseudotuberculosis: Association with Proinflammatory Cytokines in Caseous Lymphadenitis Pyogranulomas.

Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a causative agent of numerous chronic diseases, including caseous lymphadenitis (CLA) in sheep and goats, which has a zoonotic potential in humans in addition to a poor therapeutic response. In this study, out of 120 collected samples, only 12 (10%) were positive for C. pseudotuberculosis by PCR and by intraperitoneal injection of male Guinea pigs and then characterized for antimicrobial susceptibility and its genetic-relatedness by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), which showed 2-4 bands ranging from 100 to 3000 bp that can be clustered into four clusters (C1-C4). Despite the serotype biovar 1 only infecting sheep and goats, ERIC-PCR reveals intra-subtyping variation. Examination of affected LNs and organs revealed marked enlargement with either thick creamy green pus or multiple abscesses of variable sizes with a central caseated core surrounded by dense fibrous capsule. A histopathological examination revealed a central necrotic core surrounded by a peripheral mantle of mononuclear cells and a fibrous capsule. Positive immune expression of nuclear factor kappa B (NF-κB/p65) and interleukin-1β (IL-1β) and negative expression of tumor necrosis factor (TNF) in CLA is the first report to our knowledge. Conclusion: In CLA pyogranulomas, IL1β is a more crucial proinflammatory cytokine than TNF in the regulation of C. pseudotuberculosis infection, which is accompanied by marked NF-κB immunoexpression. Therefore, the NF-κB/p65 signaling pathway is involved in the activation of IL1β, and additional immunohistochemical studies are required to determine the various roles of NF-κB/p65 in the inflammatory response within CLA pyogranulomas to control this pathogen.

First Report of Potentially Pathogenic Klebsiella pneumoniae from Serotype K2 in Mollusk Tegillarca granosa and Genetic Diversity of Klebsiella pneumoniae in 14 Species of Edible Aquatic Animals.

Klebsiella pneumoniae can cause serious pneumonitis in humans. The bacterium is also the common causative agent of hospital-acquired multidrug-resistant (MDR) infections. Here we for the first time reported the genetic diversity of K. pneumoniae strains in 14 species of edible aquatic animals sampled in the summer of 2018 and 2019 in Shanghai, China. Virulence-related genes were present in the K. pneumoniae strains (n = 94), including the entB (98.9%), mrkD (85.1%), fimH (50.0%), and ybtA (14.9%) strains. Resistance to sulfamethoxazole-trimethoprim was the most prevalent (52.1%), followed by chloramphenicol (31.9%), and tetracycline (27.7%), among the strains, wherein 34.0% had MDR phenotypes. Meanwhile, most strains were tolerant to heavy metals Cu2+ (96.8%), Cr3+ (96.8%), Zn2+ (91.5%), Pb2+ (89.4%), and Hg2+ (81.9%). Remarkably, a higher abundance of the bacterium was found in bottom-dwelling aquatic animals, among which mollusk Tegillarca granosa contained K. pneumoniae 8-2-5-4 isolate from serotype K2 (ST-2026). Genome features of the potentially pathogenic isolate were characterized. The enterobacterial repetitive intergenic consensus polymerase chain reaction&amp;nbsp;(ERIC-PCR)-based genome fingerprinting classified the 94 K. pneumoniae strains into 76 ERIC genotypes with 63 singletons, demonstrating considerable genetic diversity in the strains. The findings of this study fill the gap in the risk assessment of K. pneumoniae in edible aquatic animals.

Epidemiology, Evolution of Antimicrobial Profile and Genomic Fingerprints of Pseudomonas aeruginosa before and during COVID-19: Transition from Resistance to Susceptibility.

The purpose of the study was to describe the epidemiological implication of Pseudomonas aeruginosa between 2017-2022 in a tertiary hospital from Romania, including the molecular fingerprinting of similar phenotypic strains (multidrug-resistant isolates), which would have an important health impact. The study also describes the resistance profile of P. aeruginosa before and during COVID-19, which might bring new information regarding the management of antibiotic treatments. Information regarding wards, specimen types, species, and antibiotic resistance profile of 1994 strains of Pseudomonas spp. Isolated over a period of 6 years in Mures Clinical County Hospital, Romania, was collected from the WHONET database. From 50 multidrug-resistant isolates, molecular fingerprinting was performed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) to prove the potential clonal distribution. A number of 1994 Pseudomonas spp. were isolated between 2017-2022, from which P. aeruginosa was the most frequent species, 97.39% (n = 1942). P. aeruginosa was most frequently isolated in 2017 (n = 538), with the dermatology department as the main source, mainly from pus secretion. A drop in the harvesting rate was noted in 2020 due to COVID-19 restrictions. Regarding the resistance profile, there are a few modifications. The susceptibility of P. aeruginosa to carbapenems, piperacillin-tazobactam, and amikacin suffered alterations before and during COVID-19. The molecular fingerprinting showed three P. aeruginosa clusters, including strains with 80-99% similarity.

Prevalence of classes 1 and 2 integrons in multidrug-resistant Acinetobacter baumanni isolates recovered from some aquatic environment in South Africa

The emergence of antibiotic-resistance genes (ARGs) by means of integrons in multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) has become a significant challenge in the management of infections from this pathogen. In this paper, we report on the variable region of class 1 and 2 integrons observed in MDR A. baumanni isolates recovered from rivers in the Eastern Cape Province, South Africa. Class 1 and 2 integrons with their variable regions were evaluated with polymerase chain reaction techniques followed by sequencing. Antibiotic sensitivity testing, checkerboard assay, time-kill independent assay, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) were carried out using standard microbiological techniques. A total of fifty-six (56) isolates were examined, among which 45 (79%) tested positive for class 1 integron, and 7 (12.3%) had class 2 integron. None was found to be class 3 integron positive among the isolates. The variable region contained aadA1, aadA5, and aadA2 genes, which confer resistance against streptomycin and spectinomycin, aac(6′)-Ib against amikacin/ tobramycin and dfrA17 genes against trimethoprim. The minimum inhibitory concentrations of the antimicrobials for one of the tested organisms were resistant against meropenem, colistin sodium methanesulfonate, tetracycline, ceftazidime, and ciprofloxacin (16, > 16, > 8, > 256, and 128 ug/mL respectively). The impact of colistin combined with quinolones (ciprofloxacin), with the FICIs (0.31) indicated synergistic effects against MDR A baumanni. However, when colistin was combined with meropenem and ceftazidime, additive effects with fractional inhibitory concentration (FIC) index ranging from 0.52 to 1 were observed. No antagonistic effect was evaluated among the examined isolates. ERIC-PCR analyses of A. baumanni isolates revealed significant genetic diversity, suggesting various sources of environmental contamination. We conclude that A. baumanni harbouring class 1 integrons in aquatic milieus are a significant source of ARGs and can transmit these elements to other organisms and consequently to man with significant public health implications.

Beta-lactamase determinants and molecular typing of carbapenem-resistant classic and hypervirulent Klebsiella pneumoniae clinical isolates from southwest of Iran.

This study investigated the molecular epidemiology of carbapenem-resistant classic Klebsiella pneumoniae (CR-cKp) and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) isolates in southwestern Iran. From 2019 to 2021, 136 (88.9%) cKp and 17 (11.1%) hvKp isolates were identified using biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance, beta-lactamases, and clonal relatedness of carbapenem-resistant isolates were investigated using disk diffusion, PCR, and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), respectively. The different markers of hvKp isolates were as follows: string test (35.3%, n = 6/17), magA (11.8%, n = 2/17), rmpA (11.8%, n = 2/17), rmpA2 (52.9%, n = 9/17), iucA (52.9%, n = 9/17), and peg344 (35.3%, n = 6/17). Also, 55.1% (n = 75/136) of cKp and 47.1% (n = 8/17) of hvKp isolates were CR-cKp and CR-hvKp, respectively. All CR-hvKp (100.0%, n = 8) isolates were MDR. Colistin, tetracycline, and tigecycline were the most effective antibiotics. The occurrence of beta-lactamase genes in 75 CR-cKp and 8 CR-hvKp isolates was as follows: bla NDM (41.3, 25.0%), bla IMP (4.0, 0.0%), bla VIM (8.0, 0.0%), bla GES (14.7, 25.0%), bla OXA-48-like (20.0, 0.0%), bla CTX-M (26.7, 12.5%), bla SHV (24.0, 12.5%), bla TEM (10.7, 0.0%), bla FOX (6.7, 0.0%), bla DHA (6.7, 0.0%), bla CMY (5.3, 0.0%), bla LAT (12.0, 0.0%), and bla ACT (8.0, 0.0%). ERIC-PCR showed a high diversity among isolates. In this study, the occurrence of MDR CR-hvKp isolates harboring bla NDM and bla GES was detected for the first time in southwestern Iran. To prevent the spread of CR-hvKp and reduce selection pressure, long-term surveillance and more effective treatment strategies should be implemented.

Evaluation of ERIC-PCR method for determining genetic diversity among Escherichia coli isolated from human and retail imported frozen shrimp and beef

There is a global concern and increasing reports regarding foodborne disease infections associated with consuming contaminated vegetables, seafood, meat, and poultry products. Among foodborne bacterial pathogens globally, Salmonella, Escherichia coli, and Shigella were the most frequently implicated in causing food poisoning infections in children and adults. In Saudi Arabia, the consumption rates of imported fresh fruits, vegetables, seafood, and meat products are considered high. Therefore, the development of simple PCR based DNA fingerprinting methods is essential to track the source and route of microbial contamination among imported frozen meat and seafood products. A total of 38 E. coli strains were subtyped using ERIC1R, ERIC2, and a pair combination (ERIC1R + ERIC2) to generate genomic fingerprinting. The three Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR)-based primers were generated in 26, 24, and 16 different genotypes while using ERIC1R, ERIC2, and ERIC1R + ERIC2, respectively. The Discrimination Index values obtained by ERIC1R, ERIC2, and ERIC1R + ERIC2 were 0.976, 0.965, and 0.903, respectively. ERIC1R and ERIC2 primers had the best discriminatory ability and typeability value and proved suitable for investigating genetic analysis among the population of E. coli strains. At the same time, the ERIC1R + ERIC2 primer pair has average discriminatory power and typeability value for differentiating E. coli strains. These results suggest that subtyping using ERIC1R and ERIC2 primer is a more reliable and rapid typing strategy for E. coli strains.

The Impact of Chlorine Disinfection of Hospital Wastewater on Clonal Similarity and ESBL-Production in Selected Bacteria of the Family Enterobacteriaceae.

Hospitals are regarded as ecological niches of antibiotic-resistant bacteria (ARB). ARB can spread outside the hospital environment via hospital wastewater (HWW). Therefore, HWW is often disinfected in local stations to minimize that risk. Chlorine-based treatment is the most popular method of HWW disinfection around the world, however, recent research has suggested that it can contribute to the spread of antimicrobial resistance (AMR). The aim of this study is to determine the impact of HWW disinfection on the clonal similarity of Enterobacteriaceae species and their ability to produce extended-spectrum beta-lactamases (ESBLs). The study was conducted in a hospital with a local chlorine-based disinfection station. Samples of wastewater before disinfection and samples of disinfected wastewater, collected in four research seasons, were analyzed. Bacteria potentially belonging to the Enterobacteriaceae family were isolated from HWW. The Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method was used to generate DNA fingerprints of all bacterial isolates. The isolates were phenotypically tested for the production of ESBLs. Antibiotic resistance genes (blaSHV, blaTEM, and blaOXA, blaCTX-M-1-group, blaCTX-M-2-group, blaCTX-9-group and blaCTX-M-8/25-group) were detected by PCR in strains with confirmed phenotypic ability to produce ESBLs. The ESBL+ isolates were identified by the sequencing of 16S rDNA. In the present study, the same bacterial clones were isolated from HWW before and after disinfection and HWW was sampled in different seasons. Genetic and phenotypic variations were observed in bacterial clones. ESBL+ strains were isolated significantly more often from disinfected than from non-disinfected HWW. The blaOXA gene was significantly more prevalent in isolates from disinfected than non-disinfected HWW. Enterobacter hormaechei and Klebsiella pneumoniae were the dominant species in ESBL+ strains isolated from both sampling sites. The results of this study indicate that chlorine-based disinfection promotes the survival of ESBL-producing bacteria and/or the transmission of genetic determinants of antimicrobial resistance. As a result, chlorination increases the proportion of ESBL-producing Enterobacteriaceae in disinfected wastewater. Consequently, chlorine-based disinfection practices may pose a risk to the environment and public health by accelerating the spread of antimicrobial resistance.

English

The present study aimed to determine the pathogenic Escherichia coli and Salmonella spp. and to investigate their phylogenetic relation by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) in retail chicken samples. A total of 75 samples were processed for isolation of E. coli and Salmonella spp. by classical cultural methods and isolates were confirmed by the species-specific PCR. Salmonella spp. was detected in 21.3% and E. coli was detected in 74.6% of the chicken carcasses. S. Enteritidis and S. Typhimurium were not detected in chickens by duplex PCR-based assay. O157 based on serotyping and PCR, was not detected in any of the isolates. Besides, virulence and toxin genes were not detected in any of the E. coli isolates. According to ERIC patterns, the obtained ribotypes showed that all Salmonella spp. isolates presented large genetic diversity, whereas only two (3.5%) of E. coli isolates were genetically identical. Although virulent E. coli, and pathogenic serotypes of Salmonella spp. were not detected in our study, it is thought that their high incidence should be considered as an indicator of failure to comply with hygienic conditions and lack of sanitary practices especially in slaughterhouses

Detection of carbapenem-resistant hypervirulent Klebsiella pneumoniae ST11-K64 co-producing NDM-1 and KPC-2 in a tertiary hospital in Wuhan

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) poses serious challenges to public health. Only a few sporadic reports of strains co-producing NDM-1 and KPC-2 (NDM-1-KPC-2-CR-hvKP strains) are available to date. This retrospective study investigated the clinical features, prevalence and antibiotic resistance of hvKP in a tertiary hospital in central China, and characterized an NDM-1-KPC-2-CR-hvKP strain (KP169). Clinical data were collected. Antimicrobial and virulence-associated phenotyping and genotyping, capsular serotype gene analysis and multi-locus sequence typing of hvKP isolates were performed. Whole-genome sequencing (WGS) was performed on strain KP169. Forty-five of 109 K.pneumoniae clinical isolates were hvKP. Of these, 37 originated from nosocomial infections and 24 expressed carbapenemases. Eight NDM-1-KPC-2-CR-hvKP strains were identified, and enterobacterial repetitive intergenic consensus polymerase chain reaction showed that they were clonally related. WGS revealed that strain KP169, which belongs to ST11-K64, had a single 5.5-Mb chromosome and six plasmids of 5.5-221.6kb. The blaNDM-1 gene was located on plasmid pKP169-P3, and blaKPC-2, blaSHV-12 and blaTEM-1 were located on IncFII/IncR pKP169-P2. IncHI 1/IncFIB virulence plasmid pKP169-P1 was similar to pKPC-CR-hvKP-C789 plasmid reported previously. Plasmid stability testing showed that blaKPC-2- and blaNDM-1-harbouring plasmids were maintained stably in the host. To the best of the authors' knowledge, this study identified the largest cohort, to date, of eight NDM-1-KPC-2-CR-hvKP strains, and suggests that antimicrobial stewardship and protocols to prevent transmission are needed urgently.

Clinical Characteristics and Risk Factors for Intra-Abdominal Infection with Chryseobacterium indologenes after Orthotopic Liver Transplantation.

The incidence of hospital-acquired infections caused by Chryseobacterium indologenes (C. indologenes) is increasing. This study investigated the epidemiological and clinical features of C. indologenes intra-abdominal infections in patients who underwent orthotopic liver transplantation (OLT). In this retrospective study, 53 consecutive non-replicate clinical isolates of C. indologenes were collected and identified from the OLT patients at a tertiary care university hospital in Shanghai in 2017. Genetic relatedness of the isolates was determined by enterobacterial repetitive intergenic consensus polymerase chain reaction DNA fingerprinting. Antimicrobial susceptibility of the isolates was measured using the microdilution broth method. Nosocomial clonal transmission of C. indologenes was confirmed by bacterial homology analysis. All C. indologenes isolates were resistant to β-lactams, carbapenems, quinolones, and aminoglycosides, and showed susceptibility to trimethoprim–sulfamethoxazole and minocycline. Multivariate risk modelling revealed that ≥2 bed transfers and an operation time of ≥8 h were independent risk factors for C. indologenes intra-abdominal infection after OLT. A nomogram was constructed based on the screened risk factors, which showed good concordance and accuracy. Clonal dissemination of C. indologenes in OLT patients was demonstrated and several risk factors for intra-abdominal infections were identified. Epidemiological surveillance of this organism and extensive surveillance programs are imperative worldwide.