Virulence-associated genes in faecal and clinical Escherichia coli isolates cultured from broiler chickens in Australia.

A healthy chicken's intestinal flora harbours a rich reservoir of Escherichia coli as part of the commensal microbiota. However, some strains, known as avian pathogenic E. coli (APEC), carry specific virulence genes (VGs) that enable them to invade and cause extraintestinal infections such as avian colibacillosis. Although several VG combinations have been identified, the pathogenic mechanisms associated with APEC are ill-defined. The current study screened a subset of 88 E. coli isolates selected from 237 pre-existing isolates obtained from commercial poultry flocks in Australia. The 88 isolates were selected based on their enterobacterial repetitive intergenic consensus (ERIC) and antimicrobial resistance (AMR) profiles and included 29 E. coli isolates cultured from chickens with colibacillosis (referred to as clinical E. coli or CEC) and 59 faecal E. coli (FEC) isolates cultured from clinically healthy chickens. The isolates were screened for the presence of 35 previously reported VGs. Of these, 34 were identified, with iucA not being detected. VGs focG, hlyA and sfa/foc were only detected in FEC isolates. Eight VGs had a prevalence of 90% or above in the CEC isolates. Specifically, astA (100%); feoB (96.6%); iutA, iss, ompT, iroN and hlyF (all 93.1%); and vat (89.7%). The prevalence of these were significantly lower in FEC isolates (astA 79.7%, feoB 77.9%, iutA 52.5%, iss 45.8%, ompT 50.9%, iroN 37.3%, hlyF 50.9% and vat 42.4%). The odds ratios that each of these eight VGs were more likely to be associated with CEC than FEC ranged from 7.8 to 21.9. These eight VGs may be used to better define APEC and diagnostically detect APEC in Australia. Further investigations are needed to identify the roles of these VGs in pathogenicity.

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: insights from a tertiary hospital in Southern Thailand.

Broad-spectrum ampicillin-resistant and third-generation cephalosporin-resistant Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae that have pathological features in humans, have become a global concern. This study aimed to investigate the prevalence, antimicrobial susceptibility, and molecular genetic features of extended-spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumoniae isolates in Southern Thailand. Between January and August 2021, samples (n = 199) were collected from a tertiary care hospital in Southern Thailand. ESBL and AmpC-lactamase genes were identified using multiplex polymerase chain reaction (PCR). The genetic relationship between ESBL-producing E. coli and K. pneumoniae was determined using the enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction. ESBL-producing E. coli and K. pneumoniae isolates were mostly collected from catheter urine samples of infected female patients. The ESBL production prevalence was highest in the medical wards (n = 75, 37.7%), followed by that in surgical wards (n = 64, 32.2%) and operating rooms (n = 19, 9.5%). Antimicrobial susceptibility analysis revealed that all isolates were resistant to ampicillin, cefotaxime, ceftazidime, ceftriaxone, and cefuroxime; 79.4% were resistant to ciprofloxacin; and 64.3% were resistant to trimethoprim-sulfamethoxazole. In ESBL-producing K. pneumoniae and E. coli, blaTEM (n = 57, 72.2%) and blaCTX-M (n = 61, 50.8%) genes were prominent; however, no blaVEB, blaGES, or blaPER were found in any of these isolates. Furthermore, only ESBL-producing K. pneumoniae had co-harbored blaTEM and blaSHV genes at 11.6%. The ERIC-PCR pattern of multidrug-resistant ESBL-producing strains demonstrated that the isolates were clonally related (95%). Notably, the presence of multidrug-resistant and extremely resistant ESBL producers was 83.4% and 16.6%, respectively. This study highlights the presence of blaTEM, blaCTX-M, and co-harbored genes in ESBL-producing bacterial isolates from hospitalized patients, which are associated with considerable resistance to beta-lactamase and third-generation cephalosporins. IMPORTANCE: We advocate for evidence-based guidelines and antimicrobial stewardship programs to encourage rational and appropriate antibiotic use, ultimately reducing the selection pressure for drug-resistant bacteria and lowering the likelihood of ESBL-producing bacterial infections.

Isolation, diversity and antimicrobial activity of planctomycetes from the Tejo river estuary (Portugal).

The discovery of new bioactive compounds is an invaluable aid to the development of new drugs. Strategies for finding novel molecules can focus on the exploitation of less studied organisms and ecosystems such as planctomycetes and brackish habitats. The unique cell biology of the underexplored Planctomycetota mean it is of particular interest. In this study, we aimed to isolate planctomycetes from the estuary of the Tejo river (Portugal). To reach this goal, macroalgae, water and sediments were sampled and diverse media and isolation techniques applied. Sixty-nine planctomycetal strains were brought into pure culture. An analysis of the 16S rRNA genes found that the majority of the isolates were affiliated to the genus Rhodopirellula. Putative novel taxa belonging to genera Stieleria and Rhodopirellula were also isolated and characterized morphologically. Enterobacterial repetitive intergenic consensus fingerprinting analyses showed higher diversity and different genotypes within close strains. Relevant biosynthetic gene clusters were found in most isolates and acetone extracts from representative strains exhibited mild antimicrobial activities against Escherichia coli and Staphylococcus aureus. Our work has not only enlarged the number and diversity of cultured planctomycetes but has also shown the potential for the discovery of bioactive compounds from the novel taxa.

Spoligotyping and polymerase chain reaction based mycobacterium bovis strains typing with methods (enterobacterial repetitive intergenic consensus-polymerase chain reaction, randomly amplified polymorphic dnas-polymerase chain reaction and out polymerase chain reaction).

Background: In this study, it was aimed to investigate Mycobacterium bovis strains isolated from lungs and lymph nodes of slaughtered animals on clonal level by using different methods such as spoligotyping, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNAs (RAPD-PCR) and OUT-PCR. Comparative evaluation of these methods was further conducted. Methods: A total of 38 M. bovis isolates were evaluated in the study. DNA isolation of all M. bovis strains isolated from pruvat free Löwenstein Jensen medium was done by boiling method for ERIC-PCR, RAPD-PCR, and OUT PCR. Mickle device was used for DNA isolation for spoligotyping method. Results: In 38 M. bovis isolates examined in our study, 4 different groups were determined by spoligotyping and RAPD-PCR test methods, and 5 different groups were detected in ERIC-PCR tests. In the OUT-PCR tests, the band which provides sufficient type separation was not observed. Conclusion: ERIC-PCR, RAPD-PCR, and OUT-PCR methods are easily applicable, simple, and relatively inexpensive methods for evaluating the differences between origins in the typing of M. bovis. The tests need to be evaluated in more detail with extensive studies.

Antibiotic resistance, biofilm forming ability, and clonal profiling of clinical isolates of Staphylococcus aureus from southern and northeastern India.

Background: Staphylococcus aureus is a pathogen endemic in India and sometimes deadly for patients in intensive care units. Objectives: To determine the antibiotic-resistance pattern, biofilm forming ability, and clonal type of S. aureus from isolates collected in Tamil Nadu (south) and the Mizoram (northeast) regions of India. Methods: We collected S. aureus isolates from diagnostic laboratories in Tamil Nadu and Mizoram. An antibiotic susceptibility test was performed according to Clinical Laboratory and Standards Institute methods. Antibiotic-resistant determinants such as mecA, mecC, blaZ, vanA, vanB, and vanC were confirmed by polymerase chain reaction (PCR). All isolates were further studied for biofilm forming ability. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used for clonal analysis. Results: A study of 206 clinical isolates showed 52.9% prevalence of methicillin-resistant S. aureus in Tamil Nadu and 49.4% in Mizoram. Minimum inhibitory concentration tests showed a high prevalence of 67% oxacillin resistance in isolates from Tamil Nadu and 49% in isolates from Mizoram. PCR showed 53% mecA in Tamil Nadu and 49% mecA in Mizoram. Vancomycin-intermediate resistance S. aureus (VISA) prevalence was lower in isolates from Tamil Nadu (4%) and Mizoram (5%). All methicillin-resistant S. aureus (MRSA) isolates formed biofilms. Clonal analysis revealed a genetic relatedness between the isolates. Conclusions: The prevalence of MRSA is high in the regions studied, with most of the clinical isolates being multidrug resistant. Adopting appropriate community-based preventive measures and establishing antimicrobial stewardship is highly recommended to minimize the dissemination in antibiotic resistance.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey

ABSTRACT: Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

RESUMO: Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Genomic characterization, antimicrobial resistance profiles, enterotoxin, and biofilm production of methicillin-resistant Staphylococcus aureus isolated from clinical and animal products origins in Eastern Turkey / Caracterização genômica perfis de resistência antimicrobiana, produção de enterotoxinas e biofilme Staphylococcus aureus resistente à meticilina isolado de origem clínica e de produtos de origem animal no leste da Turquia

Staphylococcus aureus is an opportunistic and ubiquitous pathogen found in the skin, nares, and mucosal membranes of mammals. Increasing resistance to antimicrobials including methicillin has become an important public concern. One hundred and eight (108) S. aureus strains isolated from a total of 572 clinical and animal products samples, were investigated for their biofilm capability, methicillin resistance, enterotoxin genes, and genetic diversity. Although only one strain isolated from raw retail was found as a strong biofilm producer, the percentage of antimicrobial resistance pattern was relatively higher. 17.59% of S. aureus strains tested in this study were resistant to cefoxitin and identified as methicillin-resistant S. aureus (MRSA) isolates. mecA and mecC harboring S. aureus strains were detected at a rate of 2.79% and 0.93%, respectively. In addition, staphylococcal enterotoxin genes including Sea, Seb, Sec, and Sed genes were found to be 18.5%, 32.4%, 6.5% and 3.7%, respectively. The phylogenetic relationship among the isolates showed relationship between joint calf and cow milk isolates. Multi locus sequence typing (MLST) revealed three different sequence types (STs) including ST84, ST829, and ST6238. These findings highlight the development and spread of MRSA strains with zoonotic potential in animals and the food chain throughout the world.

Staphylococcus aureus é um patógeno dúctil e ubíquo encontrado na pele, narinas e membranas mucosas de mamíferos. O aumento da resistência aos antimicrobianos, incluindo a meticilina, tornou-se uma importante preocupação pública. Cento e oito (108) cepas de S. aureus isoladas de um total de 572 amostras clínicas e de produtos animais foram investigadas por sua capacidade de biofilme, resistência à meticilina, genes de enterotoxinas e diversidade genética. Embora apenas uma cepa isolada do cru tenha sido encontrada como forte produtora de biofilme, a porcentagem do padrão de resistência antimicrobiana foi relativamente maior. Parte das cepas (17,59%) de S. aureus testadas neste estudo eram resistentes à cefoxitina e identificadas como isolados de MRSA. mecA e mecC abrigando cepas de S. aureus foram detectados a uma taxa de 2,79% e 0,93%, respectivamente. Além disso, verificou-se que os genes da enterotoxina estafilocócica, incluindo os genes Sea, Seb, Sec e Sed, eram 18,5%, 32,4%, 6,5% e 3,7%, respectivamente. A relação filogenética entre os isolados mostrou relação entre os isolados de bezerro e leite de vaca. A tipagem de sequência multiloco (MLST) revelou três tipos de sequência diferentes (STs), incluindo ST84, ST829 e ST6238. Essas descobertas destacam o desenvolvimento e a disseminação de cepas de MRSA com potencial zoonótico em animais e na cadeia alimentar em todo o mundo.

Clinical and Microbiological Characteristics of Recurrent Escherichia coli Bacteremia.

The causative agents of recurrent Escherichia coli bacteremia can be genetically identical or discordant, but the differences between them remain unclear. This study aimed to explore these differences, with regard to their clinical and microbiological features. Patients were recruited from a Japanese tertiary teaching hospital based on blood culture data and the incidence of recurrent E. coli bacteremia. We compared the patients' clinical and microbiological characteristics between the two groups (those with identical or discordant E. coli bacteremia) divided by the result of enterobacterial repetitive intergenic consensus PCR. Among 70 pairs of recurrent E. coli bacteremia strains, 49 pairs (70%) were genetically identical. Patients with genetically identical or discordant E. coli bacteremia were more likely to have renal failure or neoplasms, respectively. The virulence factor (VF) scores of genetically identical E. coli strains were significantly higher than those of genetically discordant strains, with the prevalence of eight VF genes being significantly higher in genetically identical E. coli strains. No significant differences were found between the two groups regarding antimicrobial susceptibility and biofilm formation potential. This study showed that genetically identical E. coli bacteremia strains have more VF genes than genetically discordant strains in recurrent E. coli bacteremia. IMPORTANCE Escherichia coli causes bloodstream infection, although not all strains are pathogenic to humans. In some cases, this infection reoccurs, and several reports have described the clinical characteristics and/or molecular microbiology of recurrent Escherichia coli bacteremia. However, these studies focused on patients with specific characteristics, and they included cases caused by microorganisms other than Escherichia coli. Hence, little is known about the pathogenicity of Escherichia coli isolated from the recurrent one. The significance of our study is in evaluating the largest cohorts to date, as no cohort studies have been conducted on this topic.

Genetic relatedness in extended-spectrum beta-lactamase-producing Escherichia coli from clinical isolates using enterobacterial repetitive intergenic consensus polymerase chain reaction.

OBJECTIVES: The aim of this study is to determine the genetic relatedness of extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli using the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) technique. METHODS: Suspected Gram-negative bacteria with their identities from the clinical samples were confirmed using Microgen GN-A-ID Kit. The double-disc synergy test was used to confirm for ESBL-producing E. coli. The susceptibility of the organisms was tested against eleven antimicrobial agents. A singleplex PCR assay was carried out targeting TEM, SHV, CTX-M, and OXA. ERIC-PCR performed, and band patterns obtained were visually evaluated. A dendrogram of the ERIC-PCR fingerprint pattern was done with the aid of DendroUPGMA using the cluster method. RESULTS: Of the 576 clinical samples collected, 23 isolates were confirmed E. coli, and all (100%) are ESBL producers. The highest antibiotic resistance rate was recorded in cefixime (95.6%), and the least was amikacin (17.4%). The predominant ESBL gene is blaTEM genes (95.6%). Gel analysis of ERIC-PCR revealed 1-6 bands. The profiles of the ERIC-PCR differentiated the 23 E. coli isolates into four ERIC cluster types. CONCLUSION: More than 80% of the isolates are sensitive to amikacin, with greater than 95% harboring blaTEM genes. Overall, ERIC obtained from the clinical specimens indicated some evidence in the genetic relatedness of the ESBL genes among E. coli isolates.

Duration of carriage of multidrug-resistant bacteria in dogs and cats in veterinary care and co-carriage with their owners.

BACKGROUND: The emergence and spread of multidrug-resistant organisms (MDROs) represent a threat to human and animal health. OBJECTIVES: To assess duration of carriage of MDROs in dogs and cats presented to veterinary clinics/hospitals in Switzerland. To estimate prevalence, duration of and risk factors for MDRO carriage in their owners and the occurrence of co-carriage in owner-pet pairs. METHODS: Prospective, longitudinal, observational study. Nasal swabs and fecal samples were collected from 50 owners of dogs and cats presented to 3 large veterinary hospitals, 1 medium-sized clinic and 1 practice. If pet or owner tested positive for a MDRO, follow-up samples were collected for up to 8 months. Methicillin-resistant (MR) Staphylococcus aureus, MR S. pseudintermedius, MR coagulase-negative staphylococci (MRCoNS), MR Macrococcus spp., cephalosporinase- and carbapenemase-producing (CP) Enterobacterales were isolated and further characterized by MALDI-TOF MS, microdilution, ß-lactam resistance gene detection, REP/ERIC-PCR, multilocus sequence typing or whole-genome sequencing. Risk factors for MDRO carriage in owners were explored based on questionnaire-derived data. RESULTS: Five out of 50 owners carried 3rd generation cephalosporin-resistant Enterobacterales (3GC-R-Ent.), and 5/50 MRCoNS. In 3 dogs and 4 cats carriage of 3GC-R-Ent. persisted for up to 136 days after discharge (median 99 days, IQR 83 days, range 36-136 days), in two cats isolates were carbapenem-resistant. Owner-pet co-carriage was not observed. No specific risk factors for MDRO carriage in owners were identified. CONCLUSIONS: After discharge from veterinary care, dogs and cats may carry 3GC-R-Ent. for prolonged time periods. Carriage of MDROs was common in owners, but pet-owner co-carriage of the same MDRO was not observed.

Molecular and Anti-Microbial Resistance (AMR) Profiling of Methicillin-Resistant Staphylococcus aureus (MRSA) from Hospital and Long-Term Care Facilities (LTCF) Environment.

To provide evidence of the cross-contamination of emerging pathogenic microbes in a local network between long-term care facilities (LTCFs) and hospitals, this study emphasizes the molecular typing, the prevalence of virulence genes, and the antibiotic resistance pattern of methicillin-resistant Staphylococcus aureus. MRSA isolates were characterized from 246 samples collected from LTCFs, medical tubes of LTCF residents, and hospital environments of two cities, Chiayi and Changhua. Species identification, molecular characterization, and drug resistance analysis were performed. Hospital environments had a higher MRSA detection rate than that of LTCF environments, where moist samples are a hotspot of MRSA habitats, including tube samples from LTCF residents. All MRSA isolates in this study carried the exfoliative toxin eta gene (100%). The majority of MRSA isolates were resistant to erythromycin (76.7%), gentamicin (60%), and ciprofloxacin (55%). The percentage of multidrug-resistant MRSA isolates was approximately 50%. The enterobacterial repetitive intergenic consensus polymerase chain reaction results showed that 18 MRSA isolates belonged to a specific cluster. This implied that genetically similar isolates were spread between hospitals and LTCFs in Changhua city. This study highlights the threat to the health of LTCFs' residents posed by hospital contact with MRSA.

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.

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 safe for consumption.

Community Structure of Protease-Producing Bacteria Cultivated From Aquaculture Systems: Potential Impact of a Tropical Environment.

Protease-producing bacteria play vital roles in degrading organic matter of aquaculture system, while the knowledge of diversity and bacterial community structure of protease-producing bacteria is limited in this system, especially in the tropical region. Herein, 1,179 cultivable protease-producing bacterial strains that belonged to Actinobacteria, Firmicutes, and Proteobacteria were isolated from tropical aquaculture systems, of which the most abundant genus was Bacillus, followed by Vibrio. The diversity and relative abundance of protease-producing bacteria in sediment were generally higher than those in water. Twenty-one genera from sediment and 16 genera from water were identified, of which Bacillus dominated by Bacillus hwajinpoensis in both and Vibrio dominated by Vibrio owensii in water were the dominant genera. The unique genera in sediment or water accounted for tiny percentage may play important roles in the stability of community structure. Eighty V. owensii isolates were clustered into four clusters (ET-1-ET-4) at 58% of similarity by ERIC-PCR (enterobacterial repetitive intergenic consensus-polymerase chain reaction), which was identified as a novel branch of V. owensii. Additionally, V. owensii strains belonged to ET-3 and ET-4 were detected in most aquaculture ponds without outbreak of epidemics, indicating that these protease-producing bacteria may be used as potential beneficial bacteria for wastewater purification. Environmental variables played important roles in shaping protease-producing bacterial diversity and community structure in aquaculture systems. In sediment, dissolved oxygen (DO), chemical oxygen demand (COD), and salinity as the main factors positively affected the distributions of dominant genus (Vibrio) and unique genera (Planococcus and Psychrobacter), whereas temperature negatively affected that of Bacillus (except B. hwajinpoensis). In water, Alteromonas as unique genus and Photobacterium were negatively affected by NO3 --N and NO2 --N, respectively, whereas pH as the main factor positively affected the distribution of Photobacterium. These findings will lay a foundation for the development of protease-producing bacterial agents for wastewater purification and the construction of an environment-friendly tropical aquaculture model.

Biofilm-Formation in Clonally Unrelated Multidrug-Resistant Acinetobacter baumannii Isolates.

This study analyzed the genotype, antibiotic resistance, and biofilm formation of Acinetobacter baumannii strains and assessed the correlation between biofilm formation, antibiotic resistance, and biofilm-related risk factors. A total of 207 non-replicate multi-drug-resistant A. baumannii strains were prospectively isolated. Phenotypic identification and antimicrobial susceptibility testing were carried out. Isolate biofilm formation ability was evaluated using the tissue culture plate (TCP), Congo red agar, and tube methods. Clonal relatedness between the strains was assessed by enterobacterial repetitive intergenic consensus-PCR genotyping. Of the 207 isolates, 52.5% originated from an intensive care unit setting, and pan resistance was observed against ceftazidime and cefepime, with elevated resistance (99-94%) to piperacillin/tazobactam, imipenem, levofloxacin, and ciprofloxacin. alongside high susceptibility to tigecycline (97.8%). The Tissue culture plate, Tube method, and Congo red agar methods revealed that 53.6%, 20.8%, and 2.7% of the strains were strong biofilm producers, respectively, while a significant correlation was observed between biofilm formation and device-originating respiratory isolates (p = 0.0009) and between biofilm formation in colonized vs. true infection isolates (p = 0.0001). No correlation was detected between antibiotic resistance and biofilm formation capacity, and the majority of isolates were clonally unrelated. These findings highlight the urgent need for implementing strict infection control measures in clinical settings.

Multidrug-resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolated from a subtropical river contaminated by nearby livestock industries.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major threat to public health that causes infections in hospitals, communities, and animal husbandry. Livestock-associated MRSA (LA-MRSA) is defined as MRSA possessing staphylococcal cassette chromosome mec (SCCmec) IV or V, both of which lacks the Panton-Valentine leukocidin (PVL) gene but has variable combinations of antimicrobial susceptibility. This study focused on Taiwan's subtropical river basin and the Puzih River, which converges from tributaries flowing through downtown and animal husbandry areas. MRSA was detected at a rate of 7.8% in the tributaries, which was higher than downstream (2.1%). The ratio of multidrug-resistant (MDR) MRSA (n = 30) to total MRSA isolates (n = 39) was 0.769, and most of the MDR MRSA isolates (66.7%, 20/30) exhibited resistance to chloramphenicol, ciprofloxacin, clindamycin, erythromycin, sulfamethoxazole-trimethoprim, and tetracycline. The number of MDR MRSA isolates in the tributaries was also higher than the downstream regions of the Puzih River. The majority of MRSA isolates (64.1%) observed in this study possessed SCCmec type IV without PVL, which is typical for LA-MRSA. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) typing aided the discrimination of resistance patterns among SCCmec types. This study highlights the threat to human health posed by the waterborne transmission of MDR LA-MRSA.

Heteroresistance to colistin in oxacillinase-producing carbapenem-resistant Acinetobacter baumannii clinical isolates from Gorgan, Northern Iran.

OBJECTIVES: Colistin resistance rates are rising globally among multidrug-resistant Gram-negative bacilli, including Acinetobacter baumannii (A. baumannii). A new type of resistance - heteroresistance - has also been reported to colistin in clinical A. baumannii isolates. This study investigated the presence of colistin heteroresistance in carbapenem-resistant A. baumannii clinical isolates. METHODS: Different clinical specimens from hospitalised patients were investigated for A. baumannii. The MICs to imipenem, meropenem and colistin were determined by broth microdilution. PCR was performed to detect OXA-type carbapenemase genes (blaOXA-23-like, blaOXA-24/40-like, blaOXA-51-like, blaOXA-58-like, and blaOXA-143-like). Heteroresistance to colistin was examined using the population analysis profiles method. Genotypic relatedness of the isolates was analysed by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). RESULTS: Overall, 71 A. baumannii isolates were recovered from clinical specimens. Of these, 27 (38.03%) and 44 (61.97%) isolates were carbapenem-susceptible and carbapenem-resistant, respectively. In addition, 67 (94.36%) isolates were susceptible to colistin, with MICs between 0.25-2 µg/mL. Among the 44 selected carbapenem-resistant colistin-susceptible isolates, the frequency of blaOXA-51-like, blaOXA-23-like and blaOXA-24/40-like genes was 100%, 77.27% and 43.18%, respectively. Nine of 44 (20.45%) isolates were characterised as colistin-heteroresistant with subpopulations growing at 6-8 µg/mL, whereas two of 44 (4.54%) presented heterogeneous subpopulations growing at up to 1 µg/mL of colistin. ERIC­PCR typing clustered A. baumannii isolates to 10 common types (CT1-CT10) containing isolates from different hospitals and 12 single types (ST1-ST12). CONCLUSIONS: A. baumannii with a colistin heteroresistance phenotype was common. This could be of great concern since colistin is often used as a last-resort drug for treating A. baumannii infections, highlighting that care is necessary with colistin monotherapy. In addition, more effective strategies and surveillance are required to confine and prevent the inter-hospital and/or intra-hospital dissemination of A. baumannii between therapeutic centres.

Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan.

BACKGROUND: Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis strains can exist in single animals. Typing techniques are required for identifying G. parasuis isolates. Different strains within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence-associated trimeric autotransporters (vtaA) gene is an indicator of virulence. The aim of this study was to characterize Taiwanese G. parasuis isolates via molecular serotyping, vtaA PCR and ERIC-PCR. METHODS: One hundred and forty-five strains were collected between November 2013 and March 2017 in Taiwan and further examined by molecular serotyping, vtaA PCR and ERIC-PCR. RESULTS: The dendrogram revealed heterogeneous genetic diversity within many clusters. Partial correlation between the ERIC-PCR clusters of different strains, serovars and lesion patterns was observed. Twelve herds (8.3%) infected with more than one strain. Group 1 vtaA positive rate reached 98.6%. DISCUSSION: This study showed the high genetic diversity of G. parasuis in Taiwan by a high discriminatory capability of ERIC-PCR. Group 1 vtaA commonly exists in G. parasuis isolates and may play important roles in the pathogenesis of Taiwanese G. parasuis isolates.

Phenotypic and molecular detection of metallo-ß-lactamase-producing Pseudomonas aeruginosa isolates from patients with burns in Tehran, Iran

Abstract INTRODUCTION: Health care-associated infections caused by metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa are a significant growing concern in patients with burns worldwide. The aims of this study were to determine the antibiotic susceptibility of and detect the presence of MBLs among P. aeruginosa isolates and assess their clonal relationship using enterobacterial repetitive intergenic consensus (ERIC)-PCR. METHODS: Non-duplicated clinical isolates (160) of P. aeruginosa were collected from patients with burns at the Motahari Hospital in Tehran, Iran. All isolates were identified using standard laboratory methods and further characterized for antimicrobial susceptibility. Any carbapenem-resistant isolates were then examined for MBL production by the E-test and MBL-encoding genes were detected by PCR. The clonal relatedness of MBL-producing isolates was assessed by ERIC-PCR. RESULTS: For multidrug-resistant isolates, the highest rates of susceptibility were observed for colistin 160 (100%), polymyxin B 160 (100%), and ceftazidime 32 (20%). In total, 69 (43.7%) isolates were identified as MBL producers. Twenty-eight (17.5%) isolates were positive for the bla VIM-1 gene followed by the bla IMP-1 (15.6%) and bla SPM-1 (5.6%) genes. ERIC-PCR revealed three separate genotypes, where type A (76.8%) was the most prevalent, followed by B (20.3%), and then C (2.9%). CONCLUSIONS: Our present study found that the bla IMP-1 and bla VIM-1 genes were present at a significant frequency and also detected the bla SPM-1 gene in P. aeruginosa isolates for the first time, highlighting the need for establishing suitable infection control measures to successfully treat patients and prevent further spread of these resistant organisms among patients with burns.

Molecular epidemiology of aminoglycoside resistance in clinical isolates of Klebsiella pneumoniae collected from Qazvin and Tehran provinces, Iran.

Production of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylases are two main resistance mechanisms against these antibiotics. This study determined the frequency of AMEs and 16 s rRNA methylase genes among aminoglycoside non-susceptible K. pneumoniae isolates and evaluated their clonal relationship by enterobacterial repetitive intergenic consensus (ERIC)-PCR. A total of 177 K. pneumoniae isolates were collected from hospitals of Qazvin and Tehran, Iran. The identification of isolates was done by standard laboratory methods and API 20E strips. Aminoglycosides susceptibility was determined by Kirby-Bauer method and AMEs and 16S rRNA methylase encoding genes were studied by PCR and sequencing methods. Clonal relatedness of isolates was assessed by ERIC-PCR method. In total, 74% of isolates were non-susceptible to the aminoglycosides used in the study among those kanamycin 110 (62.1%), tobramycin 91 (51.4%), and gentamycin 87 (49.2%) showed the highest rates of resistance whereas netilmicin and amikacin revealed high susceptibility rates of 67.8% and 61.0%, respectively. Of 130 aminoglycoside non-susceptible isolates, 91.5% were positive for the presence of aac(6')-Ib as the most dominant gene followed by aac(3)-II (78.5%), aph(3')-IIIa (14.6%), ant(4')-Ia (3.1%), and armA (7.7%) either alone or in combination. ERIC-PCR results showed 67.7% of non-susceptible isolates had different banding patterns followed by three distinct clones including A (16.2%), B (10.8%), and C (5.4%). Among those isolates carrying AMEs genes, 85 (68%) isolates belonged to independent groups and 21 (16.8%), 12 (9.6%), and 7 (5.6%) isolates belonged to groups A, B, and C, respectively, whereas 7 (70%) of 16S rRNA methylase-producing isolates belonged to independent groups. Our results revealed high prevalence of AMEs with the emergence of armA genes among the genetically unrelated resistant isolates of K. pneumonia in Iran, suggesting the need for more effective therapeutic strategies to reduce the selection pressure and better management of the patients infected with these resistant isolates.