Nalidixic Acid-resistant Isolates Research Articles

Megaplasmid Dissemination in Multidrug-Resistant Salmonella Serotypes from Backyard and Commercial Broiler Production Systems in the Southeastern United States.

Over the past decade, there has been a rise in U.S. backyard poultry ownership, raising concern for residential area antimicrobial-resistant (AMR) Salmonella contamination. This study aims to lay the groundwork to better understand the persistence of AMR Salmonella in residential broiler production systems and make comparisons with commercial systems. Ten backyard and 10 commercial farms were sampled at three time points across bird production. Both fecal (n = 10) and environmental (soil, n = 5, litter/compost, n = 5, feeder, and waterer swabs, n = 6) samples were collected at each visit on days 10, 31, and 52 of production for backyard farms and days 10, 24, and 38 of production for commercial farms. AMR Salmonella was characterized phenotypically by broth microdilution and genotypically by whole-genome sequencing. Overall, Salmonella was more prevalent in commercial farm samples (52.31%) over backyard farms (19.10%). Kentucky (sequence type (ST) 152) was the most common serotype found in both backyard and commercial farms. Multidrug-resistant (MDR, resistance to ≥3 or more antimicrobial classes) isolates were found in both production systems, while ciprofloxacin- and nalidixic acid-resistant and intermediate isolates were more prevalent in commercial (33%) than backyard samples (1%). Plasmids that have been associated with MDR were found in Kentucky and Infantis isolates, particularly IncFIB(K)_1_Kpn3 megaplasmid (Infantis). Our study emphasizes the need to understand the selection pressures in disseminating megaplasmids in MDR Salmonella in distinct broiler production systems.

Quinolone-resistant Escherichia coli at the interface between humans, poultry and their shared environment- a potential public health risk

BackgroundCommensal Escherichia coli residing in the guts of humans and animals are reservoirs of multidrug resistance (MDR) genes, including quinolone resistance genes, in humans and poultry. This study aimed to characterize quinolones resistance in E. coli recovered from poultry workers, chickens, and poultry farm/market environments in Abuja, Nigeria.MethodsThis was a cross-sectional study conducted between December 2018 and April 2019 comprising poultry workers, chickens and their poultry farm/market environments. This study characterized E. coli isolates from stool, faecal and environmental samples using antimicrobial susceptibility testing and whole-genome sequencing methods. Core-genome multilocus sequences-based phylogeny was used to determine the relatedness between quinolone-resistant E. coli isolates. Data were analyzed using descriptive statistics.ResultsOf 110 E. coli isolates, quinolone-resistant phenotypes were observed in 68.2% (n = 75) isolates. Whole-genome sequencing detected plasmid-mediated quinolone resistance (PMQR) genes in 63.6% (n = 70) isolates. The most prevalent PMQR gene detected in 56 of these 70 E. coli isolates was qnrS1, followed by qnrB19 in 14 isolates and aac(6’)-lb-cr in two isolates. Fifteen ciprofloxacin and 19 nalidixic acid-resistant isolates respectively showed double mutations in the quinolone-resistance determining regions (QRDRs) of gyrA, with single or double mutations in parC, and a single mutation in parE. The most prevalent amino-acid substitutions observed were S83L + D87N in gyrA (46.5%, n = 20), S80I in parC (51.2%, n = 22) and S458A in parE (14%, n = 6). About 2.9% (2/70) of PMQR isolates were extended-spectrum beta-lactamase (ESBL) producers while 2.9% (2/70) had plasmid-mediated colistin resistance (PMCR) genes.ConclusionsPMQR genes were prevalent in E. coli isolates recovered from healthy humans, chickens and poultry farm/market environments. PMCR genes (mcr-1.1) occurred in PMQR-positive isolates recovered from manure and drinking water originating from poultry farm/market environments. It was found that the gene encoding ESBL coexisted with qnrS-positive isolates of human and avian origin. Horizontal transfer of PMQR genes among E. coli isolates in the human-poultry-environment interface has public health implications for the spread of antimicrobial resistance. Relevant government agencies should enforce regulations to restrict the use of critically important antimicrobials in poultry production.

Multidrug-resistant Salmonella strains from food animals as a potential source for human infection in Iran

This study aimed to determine the frequency and characterization of Salmonella isolates from food-producing animals and human diarrheal samples in Shiraz, Iran. Overall, 105 Salmonella isolates were obtained from chicken meat (70/100, 70.0%), beef (19/100, 19.0%), lamb (11/100, 11.0%), and human stool (5/295, 1.7%). S. Infantis (40.9%), S. Enteritidis (29.5%), and S. Paratyphi B (8.6%) were the most prevalent serotypes. Totally 59.1% of the isolates were multidrug-resistant. High resistance to nalidixic acid (67.6%), tetracycline (62.9%), and trimethoprim-sulfamethoxazole (42.3%) were observed. Mutations in the gyrA and parC genes were detected in nalidixic acid-resistant isolates from chicken meat and human stool. No qnrA, qnrB or qnrS genes were detected. The blaTEM and blaCMY-2 genes were detected in β-lactam-resistant isolates from beef and lamb. Despite the high genetic diversity of PFGE patterns, some isolates from different sources and times showed identical PFGE patterns suggesting specific Salmonella species circulate between various sources over time.

Characteristics of Klebsiella Pneumonia St4 Coharboring Qnrb1, Aac-Ib-Cr, CTX-M-15 and SHV 11 in A Tunisian Hospital

Background: Multiresistant Klebsiella pneumonia are predominant cause of hospital acquired infection. This work describes the molecular epidemiology of these isolates in Tunisian Hospital. Methods: Between October 2010 and June 2013, 50 non-duplicated clinical K.pneumoniae were selected based on nalidixic acid (NA) resistance and were characterized. Isolates were identified using APi 20E system. Susceptibility testing was determined using the disc diffusion method and the micro dilution technique to determine the MIC of ciprofloxacin. PMQR and ESBL genes were detected by PCR and positive results were confirmed by direct sequencing of PCR products. Multilocus sequence typing (MLST) was performed to determine the genetic relationship among isolates. Conjugation and transformation were done to know if PMQR and ESBL were carried with one or two plasmids. Results: 20 PMQR harboring K.pneumoniae representing 40% of all NA resistant isolates were characterized. Among PMQR positive K.pneumoniae 13 were resistant to amoxicillin, amoxicillin/clavulanic acid, ticarcilline, piperacillin, cefaloridine, cefotaxime (CTX) and ceftazidime. The rate of resistance to gentamicin, tobramycin and amikacin were 85%, 95% and 25% respectively. Out of 20 K.pneumoniae (60%) were qnr positive (1 qnrA6 and 11 qnrB1) and (60%) were aac-Ib-cr positive. 33.3% harbored the aac-Ib-cr and qnrB1 determinants. Out of all PMQR positive strains, 65% harbored ctx-M-15 gene. It was associated to shv11 in three cases and tem1 in two cases. The predominant types were ST4 (35%) and ST15 (20%). ST 101(15%) and ST 147 (10%) come in second order. one case of each ST14,ST86,ST336 and ST307 were also observed. qnrB1, aac-Ib-cr, Ctx-m-15 can be carried with more than one plasmid in the same bacteria. Conclusion: The co-existing of different genes conferring resistance among the same and different family of antibiotics is a big threat to patient because it limits the therapeutic process. This phenomenon is a problem of concern that needs to improve the resistance surveillance of multi gene carrying K pneumonia.

Genetic diversity and multidrug resistance of phylogenic groups B2 and D in InPEC and ExPEC isolated from chickens in Central China

BackgroundAvian colibacillosis is an infectious bacterial disease caused by avian pathogenic Escherichia coli (APEC). APEC causes a wide variety of intestinal and extraintestinal infections, including InPEC and ExPEC, which result in enormous losses in the poultry industry. In this study, we investigated the prevalence of InPEC and ExPEC in Central China, and the isolates were characterized using molecular approaches and tested for virulence factors and antibiotic resistance.ResultsA total of 200 chicken-derived E. coli isolates were collected for study from 2019 and 2020. The prevalence of B2 and D phylogenic groups in the 200 chicken-derived E. coli was verified by triplex PCR, which accounted for 50.53% (48/95) and 9.52% (10/105) in ExPEC and InPEC, respectively. Additionally, multilocus sequence typing method was used to examine the genetic diversity of these E. coli isolates, which showed that the dominant STs of ExPEC included ST117 (n = 10, 20.83%), ST297 (n = 5, 10.42%), ST93 (n = 4, 8.33%), ST1426 (n = 4, 8.33%) and ST10 (n = 3, 6.25%), while the dominant ST of InPEC was ST117 (n = 2, 20%). Furthermore, antimicrobial susceptibility tests of 16 antibiotics for those strains were conducted. The result showed that more than 60% of the ExPEC and InPEC were resistant to streptomycin and nalidixic acid. Among these streptomycin resistant isolates (n = 49), 99.76% harbored aminoglycoside resistance gene strA, and 63.27% harbored strB. Among these nalidixic acid resistant isolates (n = 38), 94.74% harbored a S83L mutation in gyrA, and 44.74% harbored a D87N mutation in gyrA. Moreover, the prevalence of multidrug-resistant (MDR) in the isolates of ExPEC and InPEC was 31.25% (15/48) and 20% (2/10), respectively. Alarmingly, 8.33% (4/48) of the ExPEC and 20% (2/10) of the InPEC were extensively drug-resistant (XDR). Finally, the presence of 13 virulence-associated genes was checked in these isolates, which over 95% of the ExPEC and InPEC strains harbored irp2, feoB, fimH, ompT, ompA. 10.42% of the ExPEC and 10% of the InPEC were positive for kpsM. Only ExPEC isolates carried ibeA gene, and the rate was 4.17%. All tested strains were negative to LT and cnf genes. The carrying rate of iss and iutA were significantly different between the InPEC and ExPEC isolates (P < 0.01).ConclusionsTo the best of our knowledge, this is the first report on the highly pathogenic groups of InPEC and ExPEC in Central China. We find that 50.53% (48/95) of the ExPEC belong to the D/B2 phylogenic group. The emergence of XDR and MDR strains and potential virulence genes may indicate the complicated treatment of the infections caused by APEC. This study will improve our understanding of the prevalence and pathogenicity of APEC.

A survey of antimicrobial-resistant Escherichia coli prevalence in wild mammals in Japan using antimicrobial-containing media.

The emergence and spread of antimicrobial-resistant bacteria and resistance genes pose serious human and animal health concerns. Therefore, to control antimicrobial-resistant bacteria in the environment, the status of antimicrobial resistance of Escherichia coli in a variety of wild mammals and their prevalence were examined using antimicrobial-containing media. In total, 750 isolates were obtained from 274/366 (74.9%) wild mammals, and antimicrobial-resistant E. coli was detected in 37/750 isolates (4.9%) from 7 animal species (26/366 [7.1%] individuals). Using antimicrobial-containing media, 14 cefotaxime (CTX)- and 35 nalidixic acid-resistant isolates were obtained from 5 (1.4%) and 17 (4.6%) individuals, respectively. CTX-resistant isolates carried blaCTX-M-27, blaCTX-M-55, blaCTX-M-1, and blaCMY-2, with multiple resistance genes. Fluoroquinolone-resistant isolates had multiple mutations in the quinolone-resistance determining regions of gyrA and parC or qnrB19. Most resistant isolates exhibited resistance to multiple antimicrobials. The prevalence of antimicrobial-resistant bacteria observed in wild mammals was low; however, it is essential to elucidate the causative factors related to the low prevalence and transmission route of antimicrobial-resistant bacteria/resistance genes released from human activities to wild animals and prevent an increase in their frequency.

Molecular epidemiology and antimicrobial resistance of invasive non-typhoidal Salmonella in China, 2007-2016.

PurposeHuman infections caused by invasive non-typhoidal Salmonella (iNTS) are highly prevalent worldwide. However, data for such infections in China are scarce. This study reports the epidemiology of iNTS in China.MethodsINTS isolates were recovered from blood and other clinical specimens collected during 2007–2016 across five provinces (Shanghai, Xinjiang, Fujian, Guangxi, and Chongqing) in China. Antimicrobial susceptibility was performed using the agar dilution method and molecular epidemiology was performed using standard microbiological techniques.ResultsA total of 178 iNTS isolates were recovered from approximately 9700 patient specimens during 2007–2016. The predominant serovars were Salmonella Enteritidis (57/178, 32%), Salmonella Choleraesuis (47/178, 26.4%), and Salmonella Typhimurium (24/178, 13.5%). Up to 50 isolates (28.1%) were from patients who were ≤1 year of age, while 28 (15.7%) were from patients who were ≥60 years. Among these isolates, high rates of resistance to nalidixic acid (114/178, 64%), sulfisoxazole (59%), ciprofloxacin (15.2%), and cefotaxime (8.4%) were found. Moreover, 53.4% (95/178) exhibited multidrug resistance, and 3.9% (7/178) showed co-resistance to third-generation cephalosporins and ciprofloxacin. Steadily increasing numbers of nalidixic acid, cefotaxime, and ciprofloxacin-resistant isolates, but decreasing numbers of multidrug resistance isolates were detected during the study period. Detection of quinolone genes in 114 nalidixic acid-resistant isolates showed that 58.3% (67/114) harbored plasmid-mediated quinolone resistance (PMQR) genes [aac(6´)-Ib-cr, qnrA, qnrB, oqxAB, qepA, qnrS, and qnrD] and 98.2% (112/114) exhibited mutations in quinolone resistance determining regions [gyrA, parC, and parE]. Furthermore, we detected beta-lactamases genes in the ceftriaxone-resistant isolates. The most common were blaTEM-1 (93.3%), followed by blaCTX-M-55 (40%), blaCMY-2 (33.3%), and blaOXA-1 (33.3%). Finally, a range of pulsed-field gel electrophoresis patterns were detected among the Salmonella Enteritidis and Salmonella Typhimurium isolates.ConclusionHigh rates of multidrug resistance and steadily increasing cefotaxime and ciprofloxacin-resistant iNTS could pose a significant challenge for the effective treatment of salmonellosis in China.

Occurrence of plasmid-mediated quinolone resistance determinants among Escherichia coli strains isolated from animals in Tunisia: Specific pathovars acquired qnr genes.

The aim of this study was to characterise Escherichia coli strains harbouring plasmid-mediated quinolone resistance (PMQR) genes recovered from various samples (n = 116) from healthy and diarrhoeic animals in Tunisia. All nalidixic acid-resistant E. coli isolates were screened for the presence of PMQR genes. Isolates positive for PMQR genes were investigated by PCR for chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC, the presence of class 1 and class 2 integrons, genes encoding tetracycline and sulfonamide resistance, genes encoding virulence factors, and phylogenetic group. Genetic relationships was determined by pulsed-field gel electrophoresis (PFGE). Amongst 51 nalidixic acid-resistant isolates, 9 harboured PMQR genes (5 co-harbouredqnrS1 and qnrB1, 3 harboured qnrS1 and 1 harboured qnrB1). Two types of mutation in the QRDR of GyrA were observed: S83L and D87N (eight isolates) and S83L (one isolate). For the QRDR of ParC, the substitution S80I was observed in four isolates. A class 1 integron was found in six isolates. The tetA or tetB gene was observed in six isolates and both tetA and tetB were co-harboured by two isolates. The sul1, sul2 and sul3 genes were detected in six, four and one isolates, respectively. According to the presence of specific virulence genes, the nine strains were classified as UPEC (5), EAEC (3) and EPEC (1). Three isolates from turkey faeces were clonally related by PFGE. These findings highlight the plausible role of the avian industry as a reservoir of human pathogenic E. coli strains.

Characteristics of third-generation cephalosporin-resistant Salmonella from retail chicken meat produced by integrated broiler operations

Integrated broiler operations, which control and operate vertically through all phases of the chicken industry, have applied biosecurity and sanitation practices, housing technologies, feeding regimens, and antibiotic applications in different ways to improve food safety. The objective of this study was to compare the prevalence and antimicrobial resistance profiles of Salmonella isolates recovered from 6 different integrated broiler operations and to analyze the characteristics of extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC β-lactamase (pAmpC)-producing Salmonella isolates. Among 336 chicken meat samples, 57 were observed to be positive for Salmonella. However, the prevalence varied from 6.8% to 45.8% in chicken meat, indicating variations in Salmonella occurrence among the operations. Salmonella Albany was the dominant serovar, followed by Salmonella Virchow. In the antimicrobial resistance test, nalidixic acid-resistant isolates were the most prevalent (73.7%), followed by isolates resistant to ampicillin (49.1%) and tetracycline (42.1%). Among 14 third-generation cephalosporin-resistant isolates, 9 (64.3%) ESBL/pAmpC-producing isolates were only obtained from 2 operations: blaCTX-M-15 (n = 7) and blaCTX-M-79 (n = 1) for ESBL genes and blaCMY-2 (n = 1) for pAmpC. All ESBL/pAmpC-positive isolates exhibited high minimum inhibitory concentrations (≥128 μg/mL) of most cephalosporins and showed multidrug resistance. The transfer of ESBL/pAmpC genes was confirmed in transconjugants, which had the same genes and similar resistance patterns as those of the donor. Our findings suggest that Salmonella with resistance to third-generation cephalosporins can now be found in association with integrated broiler operations, providing data to support the development of monitoring and prevention programs for the development and spread of antimicrobial resistance in integrated broiler operations.

Transfer of Antimicrobial-Resistant Escherichia coli and Resistance Genes in a Child Care Center.

Several reports describe antimicrobial-resistance transfer among children and the community in outbreak situations, but transfer between a child and a caregiver has not been examined in child care facilities under normal circumstances. We investigated the transfer of antimicrobialresistance genes, resistant bacteria, or both among healthy children and teachers. From 2007 to 2009, 104 Escherichia coli isolates were obtained from four teachers and 38 children in a child care center. Twenty-six cephem-resistant isolates were obtained from children in 2007 and 2008. In 2009, cephem-resistant isolates were detected in children as well as a teacher. Nalidixic acid-resistant isolates from the same teacher for 3 years showed low similarity (<50%) to each other. However, an isolate from a teacher in 2007 and another from a child in 2008 showed high similarity (87%). Pulsed-field gel electrophoresis revealed 100% similarity for four isolates in 2007 and one isolate in 2008, and also similarity among seven isolates carrying the virulence gene (CNF1). This study yielded the following findings: (1) a gene for extended-spectrum β-lactamase was transferred from a child to other children and a teacher; (2) a nalidixic acid-resistant isolate was transferred from a teacher to a child; and (3) a virulent bacterium was transferred between children.

Transfer of Antimicrobial-Resistant Escherichia coli and Resistance Genes in a Child Care Center.

Several reports describe antimicrobial-resistance transfer among children and the community in outbreak situations, but transfer between a child and a care giver has not been examined in child care facilities under normal circumstances. We investigated the transfer of antimicrobial-resistance genes, resistant bacteria, or both among healthy children and teachers. From 2007 to 2009, 104 Escherichia coli isolates were obtained from four teachers and 38 children in a child care center. Twenty-six cephem-resistant isolates were obtained from children in 2007 and 2008. In 2009, cephem-resistant isolates were detected in children as well as a teacher. Nalidixic acid-resistant isolates from the same teacher for 3 years showed low similarity (<50%) to each other. However, an isolate from a teacher in 2007 and another from a child in 2008 showed high similarity (87%). Pulsed-field gel electrophoresis revealed 100% similarity for four isolates in 2007 and one isolate in 2008, and also similarity among seven isolates carrying the virulence gene (CNF1). This study yielded the following findings: (1) a gene for extended-spectrum β-lactamase was transferred from a child to other children and a teacher; (2) a nalidixic acid-resistant isolate was transferred from a teacher to a child; and (3) a virulent bacterium was transferred between children.

Molecular epidemiology and antimicrobial resistance mechanisms of Campylobacter coli from diarrhoeal patients and broiler carcasses in Belgium.

The aim of this study was to better understand the molecular epidemiology of Campylobacter coli isolated from multiple sources in Belgium, by studying the genotypic diversity and antimicrobial resistance phenotypes and resistance mechanisms of 59 C.coli isolates. Isolates from broiler carcasses and human cases were genotyped using multilocus sequence typing (MLST), porA typing, flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, and by PCR binary typing (P-BIT). Thirty-two MLST sequence types, 24 flaA types, 31 porA alleles, and 29 P-BIT types were identified among the screened isolates. Some types and alleles were shared among strains recovered from both broiler carcasses and diarrhoeal patients. Both porA typing and MLST revealed a similar discriminatory power (0.969), which was the highest discriminatory power when compared to other methods. Minimum inhibitory concentrations against seven different antibiotics (ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, tetracycline, gentamicin, and erythromycin) were analysed. Strains were most frequently resistant to tetracycline (81.4%), followed by: ciprofloxacin and nalidixic acid (76.3%); streptomycin (33.9%); erythromycin (27.1%); and chloramphenicol (3.4%). All isolates were sensitive to gentamicin. Multidrug resistance was observed in 24 of 59 C.coli isolates (40.7%). Molecular screening of antimicrobial resistance mechanisms revealed the predominance of the gyrA T86I substitution among ciprofloxacin- and nalidixic acid-resistant isolates, the tet(O) gene among tetracycline-resistant isolates and the 23S rRNA A2075G mutation among erythromycin- resistant isolates. Furthermore, some erythromycin-resistant isolates harboured a diverse array of resistance mechanisms, including the presence of ermB, 23S rRNA A2074G mutation, and point mutations the rplD and rplV ribosomal genes, and the cmeABC multidrug efflux pump genes.

The Molecular Study of Antibiotic Resistance to Quinolones in Salmonella enterica Strains Isolated in Tehran, Iran.

Introduction: Salmonella is known as one of the most important causes of gastrointestinal disease in the world. Quinolones and fluoroquinolones are used successfully in the treatment of salmonellosis particularly for infections that have become resistant to several antibiotics. But non-susceptible isolates to quinolones have been reported in several countries. The data are limited about the prevalence of quinolone-resistant isolates in our country. Therefore, this study investigated the plasmid-mediated quinolone resistance genes in Salmonella enterica isolated in Children's Medical Center in Tehran during 2014-2015.Methods and Materials: Salmonella isolates were isolated and identified using standard microbiological methods. Antibiotic susceptibility testing and screening of Salmonella strains resistant to quinolones were performed according to the CLSI guidelines. The molecular investigation was done using specific primers for detection of qnr genes including: qnrA, qnrB and qnrS, by polymerase chain reaction.Results:Overall, 92 (66.6%) strains were resistant to nalidixic acid. None of the strains showed resistance to ciprofloxacin. Out of the 92 nalidixic acid resistant strains, 52 (56.52%) harbored qnrS genes, 15 strains (16.30%) had both qnrA and qnrS genes. Two (1.1%) isolates were positive for qnrB gene. Twenty four (26.08%) nalidixic acid resistant isolates did not have any qnr qens.Conclusion:The results of this study show high prevalence of resistance to nalidixic and qnr genes in Salmonella isolates. Plasmid nature of this type of resistance poses an increased risk of dissemination of quinolone resistance between Salmonella and non-Salmonella isolates circulating in hospitals environments.

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety

ABSTRACTFoodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.

Molecular Subtyping of Salmonella enterica Serovar Typhi by Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis in India: Their Association with Antimicrobial Resistance Profiles.

Molecular subtyping and DNA sequencing-based methods, which are commonly used for discriminating Salmonella enterica serovar Typhi (S. Typhi) isolates, lead to improved molecular epidemiological investigations for prevention and control of typhoid fever. We obtained S. Typhi blood isolates (n = 66) from India during 2007-14 for molecular subtyping by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) in association with antibiotic resistance profiles. Genotypic diversity was observed more by MLVA (Simpson's index of diversity, D value = 0.997) than PFGE (D value = 0.864). Two prevalent pulsotypes containing nalidixic acid-resistant (NALR) and NALR-ciprofloxacin-resistant (CIPR) S. Typhi isolates circulated in India. Multidrug-resistant (MDR), NALR-CIPR, and most NALR isolates were found to be clonal by PFGE. MLVA could differentiate the clonal isolates. Most of the MDR and NALR-CIPR isolates showed variation in single or double VNTR loci, whereas NALR isolates varied in more than 2 loci, reflecting higher genetic diversity among the NALR isolates. Of the 6 VNTR loci, TR4,699 (D value = 0.838) and Sal02 (D value = 0.890) loci played important roles as MLVA cluster-supporting alleles. The rapid turnaround time and high-level discriminatory power of MLVA may be useful for tracking and controlling the transmission of S. Typhi isolates during epidemiological investigations.

Molecular Detection of Mutations within the Quinolone Resistance-Determining Regions in Non Typhoidal Salmonella Isolates from Malaysia

Introduction: The efficacy of chemotherapy in bacteraemia caused by non-typhoidal Salmonella (NTS) is compromised by antibiotic resistance. Objective: This study was undertaken to describe the mechanism of resistance among clinical NTS isolates. Materials & Methodology: Thirty of NTS were isolated from blood (n = 19), stool (n = 10) and bronchioalveolar lavage (BAL; n = 1) respectively. These isolates were tested for susceptibility testing by disc diffusion method against ampicillin, gentamicin, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin and ceftriaxone. Epsilometer tests (E-test) for nalidixic acid and ciprofloxacin were performed for nalidixic acid resistant isolates by disc diffusion method. DNA sequencing was carried out on six of the nalidixic acid resistant Salmonella Enteritidis isolates to identify mutations within quinolones resistance determining regions (QRDR) of gyrA, gyrB, parC and parE genes. Results: Resistance rates of NTS isolates from blood, stool, and BAL were respectively 37%, 20% and 0% for ampicillin, 79%, 40% and 0% for tetracycline, 32%, 40% and 0% for co-trimoxazole, 37%, 10% and 100% for nalidixic acid. Eight isolates were resistant to nalidixic acid and had exhibited reduced susceptibility towards ciprofloxacin by E-test. Mutation within QRDR was detected in gyrA gene (n = 6; Asp 47 → His [3], Asp 51 → Asn [1], Asp 73 → Gly [1], and Gly 48 → Asp [1]) and double mutation was detected in parE gene (n = 3; Gly 48 → Asp [3], Glu 82 → Ser [3]). Out of six isolates, three isolates were found to have both gyrA and parE gene mutations. Conclusions: There was no mutation observed in gyrB and parC gene. Mutation in gyrA gene was sufficient to induce decreased susceptibility to ciprofloxacin. Variation in amino acid sequences are novel, while detection of other gene mutation was uncommon.

Genomic analysis of Salmonella enterica serotype Paratyphi A during an outbreak in Cambodia, 2013-2015.

In 2013, an unusual increase in the number of Salmonella enterica serotype Paratyphi A (Salmonella Paratyphi A) infections was reported in patients in Phnom Penh, Cambodia, and in European, American and Japanese travellers returning from Cambodia. Epidemiological investigations did not identify a common source of exposure. To analyse the population structure and genetic diversity of these Salmonella Paratyphi A isolates, we used whole-genome sequencing on 65 isolates collected from 1999 to 2014: 55 from infections acquired in Cambodia and 10 from infections acquired in other countries in Asia, Africa and Europe. Short-read sequences from 80 published genomes from around the world and from 13 published genomes associated with an outbreak in China were also included. Pulsed-field gel electrophoresis (PFGE) was performed on a subset of isolates. Genomic analyses were found to provide much more accurate information for tracking the individual strains than PFGE. All but 2 of the 36 isolates acquired in Cambodia during 2013–2014 belonged to the same clade, C5, of lineage C. This clade has been isolated in Cambodia since at least 1999. The Chinese outbreak isolates belonged to a different clade (C4) and were resistant to nalidixic acid, whereas the Cambodian outbreak isolates displayed pan-susceptibility to antibiotics. Since 2014, the total number of cases has decreased, but there has been an increase in the frequency with which nalidixic acid-resistant C5 isolates are isolated. The frequency of these isolates should be monitored over time, because they display decreased susceptibility to ciprofloxacin, the first-choice antibiotic for treating paratyphoid fever.

Characterization of enteropathogenic Escherichia coli of clinical origin from the pediatric population in Pakistan.

Enteropathogenic Escherichia coli (EPEC) is one of the leading causes of watery diarrhea among children. In this study EPEC isolates from the pediatric population of Pakistan (2010-2012) were subjected to phylotyping, antibiotic susceptibility, extended-spectrum beta-lactamase (ESBL) profiling and evaluation of one representative strain from each panel of phylotypesin Galleria mellonella, infection model. A total of 46/225 (20.4%) stool samples were positive for EPEC. Isolates mainly belong to D phylogroup (18, 39%) followed by nontypeable (10, 22%), B1 (9, 20%), B2 (8, 17%) and A (1, 2%). High resistance was observed for ampicillin (42, 91%), erythromycin (41, 89%), cefaclor (37, 80%), trimethoprim/sulfamethoxazole (36, 78%), tetracycline (36, 78%). Among nalidixic acid resistant isolates 13 (28%) showed presence of single nucleotide polymorphism (SNP) in parC (C330-T330) whereas 1 (2%) isolate showed gyrB (A660-T660) SNP. Furthermore, 27 (59%) isolates were ESBL producers. Representative isolates of phlyotypes A and B2 showed enhance killing of G. mellonella compared to ones belonging to phylotypes B1 and D. Non-typeable EPEC strains were frequently observed. ESBL production in ESBL producers was found to be plasmid mediated. No significant association of antibiotic resistance profile with specific phylogroup of EPEC was found, however G. mellonella infection model differentiated representative phylotypes.

Characterization of Plasmid-Mediated Quinolone Resistance Determinants in High-Level Quinolone-Resistant Enterobacteriaceae Isolates from the Community: First Report of qnrD Gene in Algeria.

The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria. A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed. The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related. Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Berta ATCC 8392 and a Nalidixic Acid-Resistant Isolate of This Strain.

Salmonella enterica subspecies enterica serovar Berta has been isolated in multiple animal species and has been implicated in human disease. Here, we report a 4.7-Mbp draft genome sequence of S. enterica serovar Berta (ATCC strain 8392) and a nalidixic acid-resistant isolate derived from this strain.