Repetitive Intergenic Consensus-polymerase Chain Reaction Research Articles

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

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

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.

Phenotypic and genotypic characterization of Enterococcus spp. from yolk sac infections in broiler chicks with a focus on virulence factors

Bacterial infections of yolk sacs contribute to increased mortality of chicks, chronic infections during their rearing, or increased selection in the flock, which in turn leads to high economic losses in poultry production worldwide. The aim of this study was a phenotypic and genotypic characterization of enterococci isolated from yolk sac infections (YSI) of broiler chickens from Poland and the Netherlands. Biochemical, matrix-assisted laser desorption/ionization (MALDI)–time-of-flight (TOF) MS, and rpoA gene sequencing identification was performed. Moreover, phenotypic and genotypic characterization of virulence factors and analysis of the clonal relationship of isolates by MALDI-TOF MS and enterobacterial repetitive intergenic consensus—polymerase chain reaction (ERIC-PCR) were performed. The biochemical test identified 70 isolates as Enterococcus faecalis and 6 as Enterococcus mundtii. The results of MALDI-TOF MS were 100% concordant with those obtained by rpoA gene sequencing, and all 76 isolates were identified as E. faecalis. Differences were noted in the β-glucuronidase, β-glucosidase, α-galactosidase, phosphatase, melibiose, lactose, and raffinose tests that is going about the results of biochemical identification. None of the isolates were beta-hemolytic on blood agar in aerobic conditions, but all but one were gelatinase positive. Among biofilm-forming isolates (30/76; 39.5%), as many as 66.7% (20/30) were Polish E. faecalis strains. Most of the isolates carried virulence genes, that is gelE, ace, asa1, efaAfs, fsrA, fsrB, fsrC, cob, cpd, and ccf, but none had the hyl gene. Some isolates harbored cyl operon genes. One Polish strain (ST16) had all of the tested cyl genes and the esp gene, considered clinically important, and showed the highest biofilm-forming ability. Nearly 50% of the isolates showed close genetic relatedness in ERIC typing. In contrast with MALDI-TOF MS cluster analysis, ERIC-PCR results did not show a relationship with the origin of the strains. Using MALDI-TOF MS, 7 peaks were found in Polish and Dutch isolates, which may type them as species-specific biomarkers in E. faecalis from YSI.

Carbapenem and Colistin Resistant Klebsiella Pneumoniae ST14 and ST2096 Dominated in Two Hospitals in Turkey

The determination of clonal interactions between microorganisms is very important in epidemiological studies. In the present study, we aimed to evaluate the resistance mechanisms and genetic relationships of carbapenem and colistin resistant Klebsiella pneumoniae (K. pneumoniae) strains isolated from inpatients at two university hospitals in Turkey. A total of 38 K. pneumoniae clinical isolates were included in the study. Identification of isolates was confirmed by 16S rRNA sequencing. Antimicrobial susceptibility testing was performed with VITEK-2 system (bio-Mérieux, France). Modified Hodge test was used for the detection of carbapenemase activity in isolates. Carbapenem resistance genes (blaOXA-48, blaNDM, blaKPC, blaIMP, blaVIM) and colistin resistance genes (mcr-1, mcr-2 and mcr-3) were investigated by PCR. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequence typing (MLST) analysis were used to determine the genetic relatedness among the isolates. We detected that 58% of isolates were positive for only blaOXA-48, 5% were only positive for blaNDM, and 34% were positive for both blaOXA-48 and blaNDM. blaKPC, blaIMP, blaVIM, mcr-1, mcr-2, and mcr-3 were not detected among the isolates. Only one carbapenem resistant isolate was negative for the carbapenemase genes tested. A total of nine profiles were found by ERIC-PCR, and MLST results showed seven different sequence types-ST14, ST16, ST79, ST101, ST1543, ST2096, and ST2832. The seven STs were grouped by PHYLOVIZ Online into four clonal complexes. The most common ST was ST14 (81%) in Center 1 and ST2096 (94%) in Center 2. We determined MLST types of carbapenem and colistin resistant K. pneumoniae isolates from two different centers. Although the most common ST types were different in these centers, both ST types were clustered in CC14. To the best of our knowledge this is the first report of ST14 and ST2096 outbreaks in Turkey.

Klinik Pseudomonas aeruginosa İzolatlarında Siprofloksasin Direnci ve Direnç Mekanizmalarının Araştırılması

Pseudomonas aeruginosa is one of the most important causes of hospital infections. Although different antibiotic groups are used for the treatment of P.aeruginosa infections, quinolone groups are distinguished by the advantages of oral administration. However, in recent years, resistance against members of this group has made treatment more difficult. The aim of this study was to investigate the epidemiological relationship and possible mechanisms of resistance in ciprofloxacin resistant P. aeruginosa isolates from Ege University Hospital. The identification of P.aeruginosa bacteria isolated from clinical samples in Ege University Medical Faculty Medical Microbiology Laboratory was determined by VITEK MS automated systems by VITEK compact, antimicrobial susceptibility. The epidemiological relationships of the ciprofloxacin resistant isolates were determined by Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The presence of qnrA, qnrB, qnrS, qepA genes, the quinolone resistance genes and nfxB, mexR, the regulatory genes of the efflux pump, was determined by PCR. The phenylalanine-arginine β-naphthylamide (PAβN) assay was used to determine the activation of the efflux pump. Twenty-two isolates (26.5 %) were found resistant to ciprofloxacin. According to the ERIC-PCR results, 11 unrelated clones were detected. Ciprofloxacin minimum inhibitory concentration (MIC) values were decreased 2-64 times in 10 isolates in the presence of PAIN. No ciprofloxacin MIC change was detected in one isolate. The presence of pump regulatory genes was determined in 10 of the 11 representative isolates, while only qnrB of the genes associated with quinolone resistance was detected in seven representative isolates. qnrA, qnrS, qepA genes were not detected in any isolate. Ciprofloxacin resistant P.aeruginosa isolates are isolated from our hospital. It is noteworthy that the isolates belonging to different genetic groups are in circulation in clinics. Basic resistance mechanisms are thought to be efflux pumps and qnrB genes.

Comparison of overfeeding effects on gut physiology and microbiota in two goose breeds

To have a better understanding of how the “gut–liver axis” mediates the lipid deposition in the liver, a comparison of overfeeding influence on intestine physiology and microbiota between Gang Goose and Tianfu Meat Goose was performed in this study. After force-feeding, compared with Gang Goose, Tianfu Meat Goose had better fat storage capacity in liver (397.94 vs. 166.54 for foie gras weight (g), P < 0.05; 6.37 vs. 2.92% for the ratio of liver to body, P < 0.05; 60.01 vs. 46.64% for fat content, P < 0.05) and the less subcutaneous adipose tissue weight (1240.96 g vs. 1440.46 g, P < 0.05). After force-feeding, the digestion–absorption capacity of Tianfu Meat Goose was higher than that of Gang Goose (5.56 vs. 3.64 and 4.63 vs. 3.68 for the ratio of villus height to crypt depth in duodenum and ileum, respectively, P < 0.05; 1394.96 vs. 782.59 and 1314.76 vs. 766.17 for the invertase activity (U/mg-prot), in duodenum and ileum, respectively, P < 0.05; 6038.36 vs. 3088.29 and 4645.29 vs. 3927.61 for the activity of maltase (U/mg-prot), in duodenum and ileum, respectively, P < 0.05). Force-feeding decreased the gene expression of Escherichia coli in the ileum of Tianfu Meat Goose; force-feeding increased the number of gut microbiota Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction band in Tianfu Meat Goose and decreased the number in Gang Goose. In conclusion, compared with Gang Goose, the lipid deposition in the liver and the intestine digestion–absorption capacity and stability were higher in Tianfu Meat Goose. Thereby, Tianfu Meat Goose is the better breed for foie gras production for prolonged force-feeding; Gang Goose possesses better fat storage capacity in subcutaneous adipose tissue. However, Gang Goose has lower gut stability responding to force-feeding, so Gang Goose is suited to force-feeding in a short time to gain the body weight and subcutaneous fat as an overfed duck for roast duck.

Virulence, Resistance, and Genomic Fingerprint Traits of Vibrio cholerae Isolated from 12 Species of Aquatic Products in Shanghai, China.

Vibrio cholerae is a waterborne bacterium and can cause epidemic cholera disease worldwide. Continuous monitoring of V. cholerae contamination in aquatic products is imperative for assuring food safety. In this study, we determined virulence, antimicrobial susceptibility, heavy metal tolerance, and genomic fingerprints of 370 V. cholerae isolates recovered from 12 species of commonly consumed aquatic products collected from July to September of 2018 in Shanghai, China. Among the species, Leiocassis longirostris, Ictalurus punetaus, Ophiocephalus argus Cantor, and Pelteobagrus fulvidraco were for the first time detected for V. cholerae. Toxin genes ctxAB, tcpA, ace, and zot were absent from all the V. cholerae isolates. However, high occurrence of virulence-associated genes was detected, such as hapA (82.7%), hlyA (81.4%), rtxCABD (81.4%, 24.3%, 80.3%, and 80.8%, respectively), and tlh (80.5%). Approximately 62.2% of the 370 V. cholerae isolates exhibited resistance to streptomycin, followed by ampicillin (60.3%), rifampicin (53.8%), trimethoprim (38.4%), and sulfamethoxazole-trimethoprim (37.0%). Moreover, ∼57.6% of the isolates showed multidrug resistant phenotypes with 57 resistance profiles, which was significantly different among the 12 species (multiple antimicrobial resistance index, p < 0.001). Meanwhile, high incidence of tolerance to heavy metals Hg2+ (69.5%), Ni2+ (32.4%), and Cd2+ (30.8%) was observed among the isolates. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based fingerprinting profiles classified the 370 V. cholerae isolates into 239 different ERIC-genotypes, which demonstrated diverse genomic variation among the isolates. Overall, the results in this study meet the increasing need of food safety risk assessment of aquatic products.

Phenotypic and Genotypic Features of Klebsiella pneumoniae Harboring Carbapenemases in Egypt: OXA-48-Like Carbapenemases as an Investigated Model.

This study aimed at the characterization of carbapenem-resistant Klebsiella pneumoniae isolates focusing on typing of the blaOXA-48-like genes. Additionally, the correlation between the resistance pattern and biofilm formation capacity of the carbapenem-resistant K. pneumoniae isolates was studied. The collected isolates were assessed for their antimicrobial resistance and carbapenemases production by a modified Hodge test and inhibitor-based tests. The carbapenemases encoding genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like) were detected by PCR. Isolates harboring blaOXA-48-like genes were genotyped by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and plasmid profile analysis. The discriminatory power of the three typing methods (antibiogram, ERIC-PCR, and plasmid profile analysis) was compared by calculation of Simpson’s Diversity Index (SDI). The transferability of blaOXA-48 gene was tested by chemical transformation. The biofilm formation capacity and the prevalence of the genes encoding the fimbrial adhesins (fimH-1 and mrkD) were investigated. The isolates showed remarkable resistance to β-lactams and non-β-lactams antimicrobials. The coexistence of the investigated carbapenemases encoding genes was prevalent except for only 15 isolates. The plasmid profile analysis had the highest discriminatory power (SDI = 0.98) in comparison with ERIC-PCR (SDI = 0.89) and antibiogram (SDI = 0.78). The transferability of blaOXA-48 gene was unsuccessful. All isolates were biofilm formers with the absence of a significant correlation between the biofilm formation capacity and resistance profile. The genes fimH-1 and mrkD were prevalent among the isolates. The prevalence of carbapenemases encoding genes, especially blaOXA-48-like genes in Egyptian healthcare settings, is worrisome and necessitates further strict dissemination control measures.

Consecutive outbreaks of Burkholderia cepacia complex caused by intrinsically contaminated chlorhexidine mouthwashes

We report 2 consecutive outbreaks of the Burkholderia cepacia complex (Bcc) in an intensive care unit (ICU) and describe its characteristics and consequences. Over a 72-day period, a multidisciplinary ICU team detected 2 distinct periods of high and unusual incidence of Bcc isolates that were recovered from cultures of endotracheal aspirate. Cultures of tap water, ultrasound gel and mouthwash (opened and unopened bottles) were performed. Bcc was identified with the BD-Phoenix and MALDI-TOF MS systems, with molecular typing using the enterobacterial repetitive intergenic consensus-polymerase chain reaction technique. In both outbreak 1 (6 patients) and outbreak 2 (5 patients), the point sources of Bcc were chlorhexidine mouthwashes of 2 different brands, both of them intrinsically contaminated. All patients had a clinical diagnosis of ventilator-associated pneumonia (VAP), and 6 died. MALDI-TOF MS identified 2 species of Bcc (B. cenocepacia and B. cepacia). Enterobacterial repetitive intergenic consensus-polymerase chain reaction typing confirmed 100% genetic similarity between patient and mouthwash isolates from each period. The first outbreak was controlled in 20 days and the second in 6 days. The surveillance program for multidrug-resistant organisms, especially in high-risk patients, with the active participation of a multidisciplinary team, was crucial for success in controlling these outbreaks.

Occurrence and genetic characteristics of mcr-1-positive colistin-resistant E. coli from poultry environments in Bangladesh

ObjectivesColistin is one of the last-resort antibiotics for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. We determined the occurrence and characteristics of mcr-1-producing Escherichia coli obtained from live bird markets (LBMs), rural poultry farms (RPFs) and rural household backyard poultry environments (HBPs) in Bangladesh. MethodsWe tested 104 extended-spectrum β-lactamase (ESBL)-producing E. coli isolated during 2017–2018 from poultry sources for colistin resistance. We analysed the resistant isolates for the mcr genes and characterized mcr-positive isolates for antibiotic susceptibility, antibiotic resistance genes, transmissible plasmids and clonal diversity. ResultsOf 104 isolates, 98 (94%) had MICcolistin ≥4 μg/mL and 14 (13.5%) were positive for mcr-1, of which 10 were from LBMs (n = 10), 3 were from RPFs and 1 was from an HBP. All 14 mcr-1 E. coli were resistant to third-generation cephalosporin and tetracycline, whereas 12 were resistant to fluoroquinolone and sulfamethoxazole, 10 were resistant to aminoglycosides and 3 were resistant to nitrofurantoin. Four isolates carried conjugative mcr-1 plasmid of 23–55 MDa in size. The 55 MDa plasmid found in two isolates carried additional resistant genes including blaCTX-M-group-1 and blaTEM-1 (ESBL), qnrB (fluoroquinolone), and rmtB (aminoglycoside). These plasmids belong to the IncF family with additional replicons: HI1 and N. Enterobacterial repetitive intergenic consensus-polymerase chain reaction revealed a heterogeneous banding pattern of mcr-1-positive isolates. ConclusionWe report a 13.5% prevalence of mcr-1-positive MDR E. coli in poultry faecal samples predominantly from LBMs in Bangladesh accentuating the need for safe disposal of poultry faeces and hygiene practices among people exposed to poultry.

High Frequency of qnr Genes in Urinary Isolates of Extended-Spectrum β-Lactamase (ESBL)-producing Klebsiella pneumoniae in Tehran, Iran

Background: The emergence and spread of plasmid-mediated quinolone resistance (PMQR) among extended-spectrum beta-lactamase (ESBL)-producing isolates have become a serious threat to global public health. Objectives: The purpose of this study was to determine the presence of PMQR determinants in ESBL-producing Klebsiella pneumoniae isolates from hospitalized patients. Methods: We collected 100 strains of K. pneumoniae from patients admitted to Milad Hospital. Antimicrobial susceptibility and ESBL production were tested based on the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to determine the presence of ESBL and PMQR genes. The genetic relatedness among the strains was evaluated by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Results: We found 51% (n = 51) of the isolates as ESBL producers. Among them, the highest resistance rates were to cefepime (76.5%), amikacin (74.5%), ciprofloxacin and levofloxacin (both 70.6%), and the lowest resistance rate was to ticarcillin (96%). Among the 51 ESBL-positive isolates, blaTEM (94.1%) was the most frequent gene, followed by blaSHV (66.7%) and blaCTXM (59.9%). The frequencies of aac(6’)-Ib, qnrS, qnrD, qnrB, qnrA, and qnrC were 35.3%, 33.3%, 31.4%, 29.4%, 13.7%, and 5.9%, respectively. Coexistence of ESBL and PMQR genes was observed in 44 (86.2%) isolates. ERIC-PCR analysis revealed that ESBL producers were genetically divergent. Conclusions: Our findings indicate that PMQR genes are highly prevalent in ESBL-producing strains of K. pneumoniae in Iran. Regular detection of ESBL strains and monitoring of their antibiotic resistance patterns may help reduce the spread of these strains in hospitals.

Genetic relatedness in carbapenem-resistant isolates from clinical specimens in Ghana using ERIC-PCR technique.

AimEnterobacterial repetitive intergenic consensus (ERIC) sequence analysis is a powerful tool for epidemiological analysis of bacterial species. This study aimed to determine the genetic relatedness or variability in carbapenem-resistant isolates by species using this technique.MethodsA total of 111 non-duplicated carbapenem-resistant (CR) Gram-negative bacilli isolates from a three-year collection period (2012–2014) were investigated by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC–PCR) in four selected hospital laboratories in Ghana. The isolates were also screened for carbapenemase and extended spectrum β-lactamase genes by PCR.ResultsA proportion of 23.4% (26/111) of the genomic DNA extracts were carriers of PCR-positive carbapenemase genes, including 14.4% blaNDM-1, 7.2% blaVIM-1 and 1.8% blaOXA-48. The highest prevalence of carbapenemase genes was from non-fermenters, Acinetobacter baumannii and Pseudomonas aeruginosa. For the ESBL genes tested, 96.4% (107/111) of the CR isolates co-harboured both TEM-1 and SHV-1 genes. The ERIC-PCR gel analysis exhibited 1 to 8 bands ranging from 50 to 800 bp. Band patterns of 93 complex dissimilarities were visually distinguished from the 111 CR isolates studied, while the remaining 18 showed band similarities in pairs.ConclusionOverall, ERIC-PCR fingerprints have shown a high level of diversity among the species of Gram-negative bacterial pathogens and specimen collection sites in this study. ERIC-PCR optimisation assays may serve as a suitable genotyping tool for the assessment of genetic diversity or close relatedness of isolates that are found in clinical settings.

Molecular characteristics of qnrS1-positive Escherichia coli resistant to quinolone

Objective To analyze the molecular characteristics of qnrS-positive Escherichia coli (E.coli) strains resistant to quinolone. Methods A total of 57 qnrS1-positive clinical isolates were collected from Fujian Medical University Union Hospital. Plasmid-mediated quinolone resistance (PMQR) genes [qnrA, qnrB, qnrC, qnrD, aac(6′)-Ib-cr, qepA and oqxAB] and β-lactamase genes (blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, blaSHV and blaTEM) were detected by PCR and then sequenced. Agar dilution method was used to analyze the antimicrobial susceptibility of the qnrS1-positive strains. Phylogenetic analysis was conducted using PCR. Multilocus sequence typing (MLST) was performed for phenotyping. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was used to evaluate the genetic similarity between those isolates. Transferability of the qnrS1 genes carried by the 57 strains was examined by conjugation test with the sodiumazide-resistant E. coli J53 as the recipient strain. Mutations in the quinolone resistance-determining regions (QRDR) in those strains were analyzed by PCR. Results All of the qnrS1-positive E. coli strains showed high resistance to quinolones. PMQR genes were harbored by 14 (24.6%) isolates. Extended spectrum β-lactamases (ESBLs)-producing isolates accounted for 68.4%. Mutations in the QRDR of gyrA, gyrB, parC and parE genes were found in 56 (98.2%) strains and the most frequent point mutations were S83L (89.5%) in gyrA gene, S80I (54.4%) in parC gene and P415V (28.1%) in parE gene. The qnrS1 gene was successful transferred from 13 (22.8%) isolates to E. coli J53 by conjugation. Five plasmid incompatibility groups were detected. Phylogenetic analysis showed that there were 36 (63.2%), 13 (22.8%), 1 (1.8%) and 7 (12.3%) isolates belonging to groups A, B1, B2 and D, respectively. The 57 qnrS1-positive E. coli strains were assigned to 50 ERIC types and 39 sequence types (ST) based on the results of ERIC-PCR and MLST. Conclusions Mutations in the QRDR in E. coli strains were associated with qnrS1 gene and might play a critical role in the dissemination of quinolone-resistant bacteria. Key words: qnrS; Escherichia coli; Quinolone resistance; Plasmid-mediated quinolone resistance; Quinolone resistance-determining region

Prevalence, molecular characterization, genetic heterogeneity and antimicrobial resistance of Listeria monocytogenes associated with fish and fishery environment in Kerala, India.

The prevalence of Listeria monocytogenes in the retail fish markets of the Kerala, India was investigated by screening 227 samples comprising of marine finfish (n=97) shellfish (n=19), ready-to-cook fish products (n=47), ready-to-eat fish products (n=10), dried fish (n=11) and retail ice (n=43). The prevalence of L. monocytogenes and L. innocua was 2·7% and 17·2% respectively. Sample category wise, prevalence of L. monocytogenes was higher in marine finfish (1·8%) and retail ice (0·9%). All the L. monocytogenes isolates carried virulent genes namely inlA, inlC, inlJ, hlyA, iap, plcA, prfA genes and majority (82%) belonged to 1/2a, 3a serogroups. L. monocytogenes isolates were multidrug-resistant and showed resistance to ampicillin, penicillin, erythromycin, tetracycline and clindamycin. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) delineated 58% genetic heterogeneity among the L. monocytogenes strains. The study reports that genetic similarities of the isolates were interlinked to their serogroup and sample origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of Listeria monocytogenes, in the retail fish markets of Kerala, India was low but their relatively higher presence in marine finfish and retail ice and virulent nature of the isolates signifies food safety concerns. Moreover, multidrug-resistant nature of these isolates may potentially lead to spread of antimicrobial resistance. This study identified retail ice as a vehicle for entry of L. monocytogenes in retail fish and hence, there is a need to ensure quality of retail ice used for maintaining the cold-chain.

Virulence, antimicrobial and heavy metal tolerance, and genetic diversity of Vibrio cholerae recovered from commonly consumed freshwater fish

Vibrio cholerae is a leading waterborne pathogen worldwide. Continuous monitoring of V. cholerae contamination in aquatic products and identification of risk factors are crucial for assuring food safety. In this study, we determined the virulence, antimicrobial susceptibility, heavy metal tolerance, and genetic diversity of 400 V. cholerae isolates recovered from commonly consumed freshwater fish (Aristichthys nobilis, Carassius auratus, Ctenopharyngodon idellus, and Parabramis pekinensis) collected in July and August of 2017 in Shanghai, China. V. cholerae has not been previously detected in the half of these fish species. The results revealed an extremely low occurrence of pathogenic V. cholerae carrying the major virulence genes ctxAB (0.0%), tcpA (0.0%), ace (0.0%), and zot (0.0%). However, high incidence of virulence-associated genes was observed, including the RTX toxin gene cluster (rtxA-D) (83.0–97.0%), hlyA (87.8%), hapA (95.0%), and tlh (76.0%). Meanwhile, high percentages of resistance to antimicrobial agents streptomycin (65.3%), ampicillin (44.5%), and rifampicin (24.0%) were observed. Approximately 30.5% of the isolates displayed multidrug resistant (MDR) phenotypes with 42 resistance profiles, which were significantly different among the four fish species (MARI, P = 0.001). Additionally, tolerance of isolates to heavy metals Hg2+ (49.3%), Zn2+ (30.3%), and Pb2+ (12.0%) was observed. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)-based fingerprinting of the 400 V. cholerae isolates revealed 328 ERIC-genotypes, which demonstrated a large degree of genomic variation among the isolates. Overall, the results of this study support the need for food safety risk assessment of aquatic products.

Characterization and subtyping of Listeria monocytogenes strains from butcher shops

We characterized L. monocytogenes strains isolated from ground beef (n = 40) and butcher shop environmental (n = 99) samples before and after implementing improvement actions. Strains were serotyped and subtyped by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and ApaI-pulsed-field gel electrophoresis (PFGE) to establish their epidemiological relationships, clarify the contamination dynamics and assess the impact of improvement measures. Serotype distribution was as follows: 1/2c (57.6%), 1/2b (18.7%), 4b (12.9%), 1/2a (6.5%) and 4a/4c (4.3%). Thirteen clones were simultaneously isolated in different butcher shops; 73.5% of establishments shared the same beef supplier. Cross-contamination was detected in L. monocytogenes-positive samples from 20% of butcher shops, mostly at the evaluation stage (91.7%). Up to five strains were isolated from each butcher shop at the evaluation (37%) and verification (22%) stages. Seven persistent clones were isolated over the three-year study period. Serotypes 4b and 1/2b significantly decreased during the evaluation stage. Although part of the contamination with L. monocytogenes was introduced by the beef supplier, the pathogen spread profusely in butcher shops, suggesting the relevance of keeping good hygiene control and management of the environment to prevent the spread of L. monocytogenes in butcher shops.

Escherichia coli Bloodstream Infections in Patients at a University Hospital: Virulence Factors and Clinical Characteristics.

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates are responsible for many bloodstream infections. The aim of this study was to characterize E. coli isolated from the bloodstreams of patients (n = 48) at the University Hospital in Brazil. Epidemiological data were obtained through the analysis of medical records and laboratory tests. By PCR analysis, we investigated the presence of virulence factors (VFs), pathogenicity islands (PAIs), extended-spectrum β-lactamase (ESBL), phylogenetic classifications (A, B1, B2, C, D, E, and F) and molecular genotype by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The mortality analysis showed that 33.3% of the deaths were associated with bacteraemia due to E. coli infections; in addition, an age between 60 and 75 years (p < 0.001; OR = 6.3[2.1–18.9]) and bacteraemia with an abdominal origin (p = 0.02; OR = 5[1.2–20.5]) were risk factors for the severity of the infection. Additionally, the presence of the afa gene was associated with mortality due to E. coli bacteraemia (p = 0.027; OR = 11.4[1.5–85.7]). Immunosuppression (27.1%), intestinal diseases (25.0%) and diabetes (18.8%), were prevalent among patients, and most of the bacteraemia cases were secondary to urinary tract infections (50.0%). The serum resistance gene traT was present in 77.1% of isolates, group capsular 2 (kpsMT II) was present in 45.8% and the K5 capsule was present in 20.8% of isolates. The isolates also showed a high prevalence for the siderophore yersiniabactina (fyuA) (70.8%) and PAI IV536 (77.1%). Phylogenetic analysis showed that group B2 (45.8%) was the most prevalent, and was the phylogroup that had a higher prevalence of VFs and PAIs. However, in this study, a considerable number of isolated bacteria were classified as group B1 (18.8%) and as group E (14.6%). Eight (16.7%) isolates were resistant to third and fourth generation cephalosporin and group CTX-M-1 (CTX-M-15) was the most prevalent ESBL type. The molecular genotyping showed two clonal lineages and several isolates that were not related to each other. This study provides additional information on the epidemiological and molecular characteristics of E. coli bloodstream infections in Brazil.

Detection and Characterization of Salmonella Serotypes in the Production Chain of Two Pig Farms in Buenos Aires Province, Argentina.

The aim of the present study was to determine the prevalence of Salmonella in the pork production chain and to characterize Salmonella isolates. From 764 samples, 35 (4.6%) were positive for Salmonella spp., as determined by biochemical tests and the presence of the invA gene. From these, 2.6, 2.0, 8.8, and 8.0% corresponded to samples collected from farms, slaughterhouses, boning rooms and retail markets, respectively. Salmonella strains were classified into five serotypes and distributed as follows: S. Typhimurium in the pork production chain, S. Kentucky in farms and slaughterhouses, S. Brandenburg in slaughterhouses, S. Livingstone in farms and S. Agona in boning rooms and retail markets. Interestingly, the antimicrobial susceptibility testing indicated that all 35 Salmonella spp.-positive isolates were resistant to at least one antimicrobial agent, and 30 were multidrug-resistant (MDR) and resistant to different classes of antibiotics. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis showed clonal relatedness among strains isolated from farms, boning rooms and retail markets. The presence of antibiotic-resistant Salmonella in food poses a potential health hazard to consumers.

Epidemiology of Haemophilus parasuis isolates from pigs in China using serotyping, antimicrobial susceptibility, biofilm formation and ERIC-PCR genotyping.

BackgroundHaemophilus parasuis is a commensal organism of the upper respiratory tract of healthy pigs and causes high morbidity and mortality in piglets. The aim of this study was to investigate the epidemiology of H. parasuis in China from 2014 to 2017.MethodsWe characterized 143 H. parasuis isolates by serotyping, antimicrobial susceptibility, biofilm formation and with enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays.ResultsSerotyping revealed serovar 5 as the most prevalent (26.6%) followed by serovars 4 (22.4%), 7 (9.1 %), 13 (6.3%), 12 (5.6 %), and non-typeable (8.4%). In a panel of 23 antimicrobials, the minimum inhibitory concentration 50% (MIC50) were in the range of 0.25–16 μg/mL and MIC90 were 2–>512 μg/mL. A total of 99 isolates of H. parasuis (69.2%) were able to form biofilms and 59.6% (59/99) performed weak biofilm-forming ability. ERIC-PCR revealed a very heterogeneous pattern with 87 clusters.DiscussionThese H. parasuis isolates showed a high serovar and genotypic lineage diversity, different abilities to form biofilms and a high degree of genetic diversity. Biofilm formation was related to antimicrobial susceptibility but there were no statistically significant associations between the antimicrobial susceptibility and either the serovars or the ERIC-PCR clusters. This study showed a high prevalence of high-MIC H. parasuis strains and suggests the need for a continuous surveillance of clinical isolates of H. parasuis.

Fingerprinting and genetic relationship among Escherichia coli strains producing B. Lactamase collected from urine using ERIC/PCR Assay

This study was conducted on 50 isolates recovered from urine samples of patients with urinary infections admitted to Azadi teaching Hospital/Dohuk City. These isolates were identified and characterized in previous study as E. coli producing extended spectrum beta lactamase enzymes, all isolates were subjected to Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC PCR) assay for estimation the fingerprinting and genetic relationship among these isolates. They were successfully producing multiple amplicons ranging from 2-12 bands with different molecular weights ranging from 5212- 213bp. The most frequent band was 724 which was amplified by 31 strains, and the least frequent were 3224, 2424, 1472 and 531bp. observed in only one strain. From the result of dendogram analysis revealed that all isolates were assigned into two main clusters, each cluster was further classified into two subgroups. Similar genotype may indicate clonal spreading of bacteria in hospital wards.