Coexistence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes among clinical Pseudomonas aeruginosa isolates in Egypt.

BACKGROUND: Data about the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) production in P. aeruginosa compared to the Enterobacteriaceae family is limited. The availability of limited therapeutic options raises alarming concerns about the treatment of multidrug-resistant P. aeruginosa. This study aimed to assess the presence of PMQR and ESBL genes among P. aeruginosa strains. METHODS: Fifty-six P. aeruginosa strains were isolated from 330 patients with different clinical infections. Phenotypically fluoroquinolone-resistant isolates were tested by PCR for the presence of six PMQR genes. Then, blaTEM, blaSHV, and blaCTX-M type ESBL genes were screened to study the co-existence of different resistance determinants. RESULTS: Overall, 22/56 (39.3%) of the studied P. aeruginosa isolates were phenotypically resistant to fluoroquinolones. PMQR-producing P. aeruginosa isolates were identified in 20 isolates (90.9%). The acc(6')-Ib-cr was the most prevalent PMQR gene (77.3%). The qnr genes occurred in 72.7%, with the predominance of the qnrA gene at 54.5%, followed by the qnrS gene at 27.3%, then qnrB and qnrC at 22.7%. The qepA was not detected in any isolate. The acc(6')-Ib-cr was associated with qnr genes in 65% of positive PMQR isolates. Significant differences between the fluoroquinolone-resistant and fluoroquinolone-susceptible isolates in terms of the antibiotic resistance rates of amikacin, imipenem, and cefepime (P value < 0.0001) were found. The ESBL genes were detected in 52% of cephalosporin-resistant P. aeruginosa isolates. The most frequent ESBL gene was blaCTX-M (76.9%), followed by blaTEM (46.2%). No isolates carried the blaSHV gene. The acc(6')-Ib-cr gene showed the highest association with ESBL genes, followed by the qnrA gene. The correlation matrix of the detected PMQR and ESBL genes indicated overall positive correlations. The strongest and most highly significant correlation was between qnrA and acc(6')-Ib-cr (r = 0.602) and between qnrA and blaCTX-M (r = 0.519). CONCLUSION: A high prevalence of PMQR genes among the phenotypic fluoroquinolone-resistant P. aeruginosa isolates was detected, with the co-carriage of different PMQR genes. The most frequent PMQR was the acc(6')-Ib-cr gene. Co-existence between PMQR and ESBL genes was found, with 75% of PMQR-positive isolates carrying at least one ESBL gene. A high and significant correlation between the ESBL and PMQR genes was detected.

Genetic insights of antibiotic resistance, pathogenicity (virulence) and phylogenetic relationship of Escherichia coli strains isolated from livestock, poultry and their handlers - a one health snapshot.

BACKGROUND: Pathogenic and non-pathogenic strains of Escherichia coli harbouring antibiotic resistance genes (ARGs) from any source (clinical samples, animal settings, or environment) might be transmitted and contribute to the spread and increase of antibiotic resistance in the biosphere. The goal of this study was to investigate the genome to decipher the repertoire of ARGs, virulence genes carried by E. coli strains isolated from livestock, poultry, and their handlers (humans), and then unveil the genetic relatedness between the strains. METHODS: Whole genome sequencing was done to investigate the genetic makeup of E. coli isolates (n = 20) [swine (n = 2), cattle (n = 2), sheep (n = 4), poultry (n = 7), and animal handlers (n = 5)] from southern India. The detection of resistome, virulome, biofilm forming genes, mobile genetic elements (MGE), followed by multilocus sequence typing (MLST) and phylogenetic analyses, were performed. RESULTS: E. coli strains were found to be multi drug resistant, with a resistome encompassing > 20 ARGs, the virulome-17-22 genes, and > 20 key biofilm genes. MGE analysis showed four E. coli isolates (host: poultry, swine and cattle) harbouring composite transposons with ARGs/virulence genes (blaTEM, dfr, qnr/nleB, tir, eae,and esp) with the potential for horizontal transfer. MLST analyses revealed the presence of ST937 and ST3107 in both livestock/poultry and their handlers. Phylogenomic analyses with global E. coli isolates (human/livestock/poultry hosts) showed close relatedness with strains originating from different parts of the world (the United States, China, etc.). CONCLUSION: The current study emphasizes the circulation of strains of pathogenic sequence types of clinical importance, carrying a diverse repertoire of genes associated with antibiotic resistance, biofilm formation and virulence properties in animal settings, necessitating immediate mitigation measures to reduce the risk of spread across the biosphere.

Prevalence and characterization of quinolone resistance and integrons in clinical Gram-negative isolates from Gaza strip, Palestine.

BACKGROUND: Gram-negative bacteria with quinolone resistance and extended-spectrum beta-lactamases (ESBLs) present significant treatment challenges. This study evaluated the prevalence and characteristics of quinolone resistance in Gram-negative strains, investigating the relationship between plasmid-mediated quinolone resistance (PMQR), ESBLs, and integrons. METHODS AND RESULTS: We collected 146 Gram-negative isolates from patients in three Palestinian hospitals. For quinolone resistance isolates, the presence and characterization of PMQR, ß-lactamase genes and integrons were studied by PCR and sequencing. Out of 146 clinical isolates, 64 (43.8%) were resistant to quinolones, with 62 (97%) being multidrug-resistant (MDR) and 33 (51.5%) ESBL-producers. PMQR-encoding genes were present in 45 (70.3%) isolates, including aac(6')-Ib-cr (26.6%), qnrA (18.8%), qnrS1 (20.8%), and qnrB (6.4%). BlaCTX-M genes were detected in 50% (32/64) of isolates, with blaCTX-M-15 being the most common. BlaTEM-1, blaSHV-1 and blaVIM genes were found in 13, 6, and 4 isolates, respectively. Class I integrons were found in 31/64 (48%) of isolates, with 14 containing gene cassettes conferring resistance to trimethoprim (dhfr17, dfrA12, dfrA1) and aminoglycosides resistance genes (aadA1, aadA2, aadA5, and aadA6). CONCLUSIONS: This study found a high rate of quinolone resistance, ESBL and integrons in clinical Gram-negative isolates from our hospitals. Urgent measures are crucial, including implementing an antimicrobial resistance surveillance system, to control and continuously monitor the development of antimicrobial resistance.

Molecular characterization of extended-spectrum ß-lactamase-producing Escherichia coli isolated from lower respiratory tract samples between 2002 and 2019 in the Central Slovenia region.

BACKGROUND: Antibiotic resistance is one of the most serious global health problems and threatens the effective treatment of bacterial infections. Of greatest concern are infections caused by extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC). The aim of our study was to evaluate the prevalence and molecular characteristics of ESBL-EC isolated over an 18-year pre-COVID period from lower respiratory tract (LRT) samples collected from selected Slovenian hospitals. OBJECTIVES AND METHODS: All isolates were identified by MALDI-TOF and phenotypically confirmed as ESBLs by a disk diffusion assay. Using a PCR approach, 487 non-repetitive isolates were assigned to phylogroups, sequence type groups, and clonal groups. Isolates were also screened for virulence-associated genes (VAGs) and antimicrobial resistance genes. RESULTS: The prevalence of ESBL-EC isolates from LRT in a large university hospital was low (1.4%) in 2005 and increased to 10.8% by 2019. The resistance profile of 487 non-repetitive isolates included in the study showed a high frequency of group 1 blaCTX-M (77.4%; n = 377), blaTEM (54.4%; n = 265) and aac(6')-Ib-cr (52%; n = 253) genes and a low proportion of blaSHV and qnr genes. Isolates were predominantly assigned to phylogroup B2 (73.1%; n = 356), which was significantly associated with clonal group ST131. The ST131 group accounted for 67.6% (n = 329) of all isolates and had a higher number of virulence factor genes than the non-ST131 group. The virulence gene profile of ST131 was consistent with that of other extraintestinal pathogenic E. coli (ExPEC) strains and was significantly associated with ten of sixteen virulence factor genes tested. Using ERIC-PCR fingerprinting, isolates with the same ERIC-profile in samples from different patients, and at different locations and sampling dates were confirmed, indicating the presence of "hospital-adapted" strains. CONCLUSION: Our results suggest that the ESBL-EC isolates from LRT do not represent a specific pathotype, but rather resemble other ExPEC isolates, and may be adapted to the hospital environment. To our knowledge, this is the first study of ESBL-EC isolated from LRT samples collected over a long period of time.

Analyzing the correlation between quinolone-resistant Escherichia coli resistance rates and climate factors: A comprehensive analysis across 31 Chinese provinces.

BACKGROUND: The increasing problem of bacterial resistance, particularly with quinolone-resistant Escherichia coli (QnR eco) poses a serious global health issue. METHODS: We collected data on QnR eco resistance rates and detection frequencies from 2014 to 2021 via the China Antimicrobial Resistance Surveillance System, complemented by meteorological and socioeconomic data from the China Statistical Yearbook and the China Meteorological Data Service Centre (CMDC). Comprehensive nonparametric testing and multivariate regression models were used in the analysis. RESULT: Our analysis revealed significant regional differences in QnR eco resistance and detection rates across China. Along the Hu Huanyong Line, resistance rates varied markedly: 49.35 in the northwest, 54.40 on the line, and 52.30 in the southeast (P = 0.001). Detection rates also showed significant geographical variation, with notable differences between regions (P < 0.001). Climate types influenced these rates, with significant variability observed across different climates (P < 0.001). Our predictive model for resistance rates, integrating climate and healthcare factors, explained 64.1% of the variance (adjusted R-squared = 0.641). For detection rates, the model accounted for 19.2% of the variance, highlighting the impact of environmental and healthcare influences. CONCLUSION: The study found higher resistance rates in warmer, monsoon climates and areas with more public health facilities, but lower rates in cooler, mountainous, or continental climates with more rainfall. This highlights the strong impact of climate on antibiotic resistance. Meanwhile, the predictive model effectively forecasts these resistance rates using China's diverse climate data. This is crucial for public health strategies and helps policymakers and healthcare practitioners tailor their approaches to antibiotic resistance based on local environmental conditions. These insights emphasize the importance of considering regional climates in managing antibiotic resistance.

Occurrence and characterization of plasmid-encoded qnr genes in quinolone-resistant bacteria across diverse aquatic environments in southern Ontario.

Antimicrobial resistance is an ever-increasing threat. The widespread usage of ciprofloxacin has led to the manifestation of resistance due to chromosomal mutations or the acquisition of plasmid-mediated quinolone resistance (PMQR) traits. Some particular PMQR traits, qnr genes, have been identified globally in clinical and environmental isolates. This study aimed to determine the prevalence of ciprofloxacin-resistant bacteria in aquatic environments in southern Ontario and investigate the extent of dissemination of ciprofloxacin resistance traits among the bacterial communities. We surveyed the prevalence of plasmid encoding qnr genes using a multiplex PCR assay of associated PMQR genes, qnrA, qnrB, and qnrS, on 202 isolates. Despite the absence of significant impacts on minimum inhibitory concentration levels, the presence of qnr genes correlates with heightened resistance to quinolones and nalidixic acid in some isolates. Taxonomic analysis highlights distinct differences in the composition and diversity of ciprofloxacin-sensitive (CipS) and ciprofloxacin-resistant (CipR) populations, with Proteobacteria dominating both groups. Importantly, CipR populations exhibit lower genetic diversity but higher prevalence of multiple antibiotic resistances, suggesting co-selection mechanisms. Co-occurrence analysis highlights significant associations between ciprofloxacin resistance and other antibiotic resistances, implicating complex genetic linkages. The results of our study signified the critical role of environmental monitoring in public health.

Plasmid-Mediated Fluoroquinolone Resistance among Enterobacterales in Africa: Systematic Review.

INTRODUCTION: According to the World Health Organization, antimicrobial resistance (AMR) is a silent global pandemic that plagues everyone. It makes therapy of infectious diseases more difficult and eventually increases morbidity and mortality. AIM: The purpose of this work is to examine existing data on plasmid-mediated quinolone resistance (PMQR), to assess the prevalence of PMQR genes in Enterobacterales, and to determine any knowledge gaps from sub-Saharan Africa. METHODOLOGY: The Preferred Reporting Items of Systematic Reviews and Meta-analyses (PRISMA) standard was followed when conducting this systematic review. The main internet databases examined for pertinent publications were PubMed, Google Scholar, and Ajol. A set of qualifying criteria were used to evaluate the qualified articles. Using the eligibility criteria, 56 full-text articles were chosen for screening. RESULT: Thirty-two (32) articles with the majority originating from West and North Africa and only one article reporting a study carried out in Central Africa were selected for this review. Escherichia coli and Ciprofloxacin were the most reported Enterobacterales and Quinolone respectively. The PMQR genes include qnr (qnrA,qnrB, qnrC, qnrD, and qnrS), aac (6') Ib, aac (6') Ib-cr, oqxAB and qepA gene. The most prevalent PMQR gene is the aac (6') Ib-cr gene (32%) followed by qnrS (26%). CONCLUSION: This study highlighted the requirement for an efficient antimicrobial resistance surveillance system in the continent and revealed a significant incidence of PMQR genes.

INTRODUCTION: Selon l'Organisation mondiale de la santé, la résistance aux antimicrobiens (RAM) est une pandémie mondiale silencieuse qui touche tout le monde. Elle rend le traitement des maladies infectieuses plus difficile et finit par augmenter la morbidité et la mortalité. OBJECTIF: L'objectif de ce travail est d'examiner les données existantes sur la résistance plasmidique aux quinolones (PMQR), d'évaluer la prévalence des gènes PMQR chez les Enterobacterales et de déterminer d'éventuelles lacunes de connaissances en Afrique subsaharienne. MÉTHODOLOGIE: La norme Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) a été suivie lors de la réalisation de cette revue systématique. Les principales bases de données Internet examinées pour des publications pertinentes étaient PubMed, Google Scholar et Ajol. Un ensemble de critères d'admissibilité a été utilisé pour évaluer les articles qualifiés. En utilisant les critères d'éligibilité, 56 articles en texte intégral ont été choisis pour le dépistage. RÉSULTAT: Trente-deux (32) articles, dont la majorité provient d'Afrique de l'Ouest et du Nord, et un seul article rapportant une étude menée en Afrique centrale, ont été sélectionnés pour cette revue. Escherichia coli et la ciprofloxacine étaient les Enterobacterales et les quinolones les plus signalées respectivement. Les gènes PMQR comprennent les gènes qnr (qnrA, qnrB, qnrC, qnrD et qnrS), aac (6 ') Ib, aac (6 ') Ib-cr, oqxAB et qepA. Le gène PMQR le plus prévalent est le gène aac (6 ') Ib-cr (32 %), suivi de qnrS (26 %). CONCLUSION: Cette étude a souligné la nécessité d'un système efficace de surveillance de la résistance aux antimicrobiens sur le continen`t et a révélé une incidence significative des gènes PMQR. MOTS-CLÉS: Enterobacterales, Escherichia coli, Quinolone, Ciprofloxacine, PMQR, "aac(6')-Ib", "aac(6')-Ib-cr", "qnr", "qepA", "oqxAB", "résistance aux antibiotiques".

Resistance in Enteric Shigella and nontyphoidal Salmonella : emerging concepts.

PURPOSE OF REVIEW: The emergence of globally resistant enteric Shigella and nontyphoidal Salmonella strains (NTS) has limited the selection of effective drugs, which has become a major challenge for the treatment of infections. The purpose of this review is to provide the current opinion on the antimicrobial-resistant enteric Shigella and nontyphoidal Salmonella . RECENT FINDINGS: Enteric Shigella and NTS are resistant to almost all classes of antimicrobials in recent years. Those with co-resistance to ciprofloxacin, azithromycin and ceftriaxone, the first-line antibiotics for the treatment of infectious diarrhoea have emerged worldwide. Some of them have caused interregional and international spread by travel, trade, MSM, and polluted water sources. Several strains have even developed resistance to colistin, the last-resort antibiotic used for treatment of multidrug-resistant Gram-negative bacteria infections. SUMMARY: The drug resistance of enteric Shigella and NTS is largely driven by the use of antibiotics and horizontal gene transfer of mobile genetic elements. These two species show various drug resistance patterns in different regions and serotypes. Hence treatment decisions for Shigella and Salmonella infections need to take into consideration prevalent antimicrobial drug resistance patterns. It is worth noting that the resistance genes such as blaCTX,mph, ermB , qnr and mcr , which can cause resistance to ciprofloxacin, cephalosporin, azithromycin and colistin are widespread because of transmission by IncFII, IncI1, IncI2 and IncB/O/K/Z plasmids. Therefore, continuous global monitoring of resistance in Shigella and Salmonella is imperative.

ESBL-producing Vibrio vulnificus and V. alginolyticus harbour a plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

In recent years, trade liberalisation has led to the spread of antibiotic-resistant bacteria (ARB) in food products. Because ARB has reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern. Here, we report the complete genome sequences of ESBL-producing Vibrio vulnificus and V. alginolyticus strains harbouring a plasmid isolated from imported seafood. First, V. vulnificus and V. alginolyticus were isolated from purchased frozen and thawed Litopenaeus vannamei shrimp, and genome extraction and sequencing were performed. Hybrid genome assemblies were performed using Unicycler and annotated using DFAST. Then genome analysis was performed using BRIG. Plasmid comparisons showed that the plasmids carried by both Vibrios are remarkably similar and encode the same antibiotic-resistance genes. The 270-310 kb region specific to both Vibrios were isolated in this study and encodes the antibiotic-resistance genes blaCTX-M and qnr. Furthermore, the mobile genetic factors ISEc9, ISVch4, and ISVpa4 are located upstream and downstream of these genes. This is the first report of ESBL-producing V. vulnificus and V. alginolyticus harbouring a common plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

Quinolone resistance and biofilm formation capability of uropathogenic Escherichia coli isolates from an Iranian inpatients' population.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major pathogen of the urinary tract infection (UTI), and biofilm formation is crucial as it facilitates the colonization in the urinary tract. We aimed to investigate the antibiotic susceptibility pattern, biofilm formation capability, distribution of quinolone resistance genes, and phylogenetic groups among UPEC isolates from an Iranian inpatients' community. METHODS AND RESULTS: A collection of 126 UPEC obtained from hospitalized patients with symptomatic UTI at 3 teaching hospitals during 2016 were included. Antibiogram of all isolates against quinolone and fluoroquinolones was performed using the disk diffusion method. Phylogenetic groups and qnr A, B, and S genes were assessed by PCR. Susceptibility pattern showed that more than 50% and 81% of the isolates were resistant to fluoroquinolones and quinolones, correspondingly. The frequency of qnrS and qnrB genes was 22% and 13.5%, correspondingly. Our result indicated no significant association between the presence of fluoroquinolone genes and antibiotic resistance to them. The frequent common phylogroup was B2 (84.1%), followed by D (10.3%), A (3.2%) and B1 (2.4%) groups. Indeed, 80.2% of the isolates were biofilm producers, so that 42.1%, 16.7% and 21.4% of them were classified as weak, moderate and strong producers, respectively. CONCLUSIONS: Our results showed considerable fluoroquinolone and quinolone resistance among UPEC along with a remarkable rate of biofilm-producing isolates from symptomatic hospitalized patients, making them a serious health concern in the region. This survey highlights the need for awareness on quinolone resistance and careful prescription of them by physicians.

Regulation Transcriptional of Antibiotic Resistance Genes (ARGs) in Bacteria Isolated from WWTP.

The incidence of antibiotics and transcriptional regulation of ARGs in isolated bacteria from wastewater needs to be explored. By HPLC, in samples of untreated wastewater, ampicillin (49.74 ± 5.70 µg/mL), chloramphenicol (0.60 ± 0.03 µg/mL), tylosin (72.95 ± 2.03 µg/mL), and oxytetracycline (0.22 ± 0.01 µg/mL) was determined. Through metagenomic analysis identified 58 bacterial species belonging to 9 phyla and at least 14 species have shown resistance to a variety of antibiotics. Twenty-two bacterial isolates were proved to be resistant to fifteen antibiotics of new generation and used in medical research to combat infectious diseases. Fourteen strains were shown to harbor plasmids in size ranges of 2-5 Kb, 6-10 Kb and plasmids with size greater than 10 Kb. By quantitative PCR it was possible to identify genes sul, qnr, cat1, aadA1, and sat-1 gene were shown to be present in gDNA samples from treated and untreated samples of wastewater and by relative expression analysis, differential expression of cat1, ermB, act, and tetA genes was demonstrated in strains that showed identity with Escherichia coli, Bacteroides fragilis, and Salmonella thyphi, and that were stressed with different concentrations of antibiotics. The presence of ARGs in untreated water samples, as well as in bacterial isolates, was indicative that in these habitats there are microorganisms that can resist ß-lactams, aminoglycosides, tetracyclines, sulfonamides, and quinolones.

High Prevalence of Plasmid-Mediated Quinolone Resistance among ESBL/AmpC-Producing Enterobacterales from Free-Living Birds in Poland.

In this study, we investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) in extended-spectrum ß-lactamase- (ESBL) and/or AmpC-type ß-lactamase-producing Enterobacterales isolates from free-living birds in Poland. The prevalence of the qnrB19 gene was 63%, and the distribution of isolates in terms of bacterial species was as follows: 67% (22/33) corresponded to Escherichia coli, 83% (5/6) to Rahnella aquatilis, 44% (4/9) to Enterobacter cloacae and 33% (1/3) to Klebsiella pneumoniae. The qnrB19 gene was also found in a single isolate of Citrobacter freundii. The molecular characteristics of qnrB19-positive isolates pointed to extended-spectrum beta lactamase CTX-M as the most prevalent one (89%) followed by TEM (47%), AmpC (37%) and SHV (16%). This study demonstrates the widespread occurrence of PMQR-positive and ESBL/AmpC-producing Enterobacterales isolates in fecal samples from wild birds. In this work, plasmid pAM1 isolated from Escherichia coli strain SN25556 was completely sequenced. This plasmid is 3191 nucleotides long and carries the qnrB19 gene, which mediates decreased susceptibility to quinolones. It shares extensive homology with other previously described small qnrB19-harboring plasmids. The nucleotide sequence of pAM1 showed a variable region flanked by an oriT locus and a Xer recombination site. The presence of a putative recombination site was detected, suggesting that interplasmid recombination events might have played a role in the development of pAM1. Our results highlight the broad geographical spread of ColE-type Qnr resistance plasmids in clinical and environmental isolates of Enterobacterales. As expected from the results of phenotypic susceptibility testing, no resistance genes other than qnrB19 were identified.

Campylobacter jejuni and Campylobacter coli in human stool samples: antibiotic resistance profiles, putative virulence determinants and molecular characterization of the isolates.

Campylobacters, especially C. jejuni and C. coli, have become one of the leading causes of acute gastroenteritis in humans worldwide in recent years. We aimed to investigate the presence, antimicrobial resistance, putative virulence genes, and molecular characterization of C. jejuni and C. coli recovered from human acute gastroenteritis cases in the study. In the study, suspected Campylobacter spp. isolates were obtained in 43 (5%) feces samples collected from a total of 850 patients who applied to the Erciyes University Medical Faculty acute clinic between March 2019 and February 2020. As a result of the phenotypic tests, these isolates were determined to be Campylobacter spp. According to the multiplex PCR, 33 of 43 Campylobacter spp. isolates were identified as C. jejuni (76%) and ten isolates were as C. coli (24%). In the disc diffusion test, the highest resistance rate was found in the trimethoprim/sulfamethoxazole (90.1%) and ciprofloxacin (90.1%), and the lowest rate was found in the amoxicillin-clavulanic acid (9.3%). In Campylobacter spp. isolates, the virulence genes cdtA, virB11, cdtB, cadF, iam, ceu, and flaA were found to be positive at rates of 26 (60%), 28 (65.6%), 13 (30%), 4 (9%), 27 (62%), 17 (39%), and 7 (16%), respectively. However, the cdtC gene was not detected in any of the isolates. The study searched tetO gene to examine the genetic aspect of tetracycline resistance, which was found in all Campylobacter spp. isolates. In the PCR reactions to investigate A2074C and A2075G mutations of macrolide resistance, it was determined as 7 (16%) and 21 (48%) of the isolates. To detect quinolone resistance, the rates of quinolone resistance-determining regions (QRDR) were 20 (45.4%) and the gyrA gene mutations in the mismatch amplification mutation assay PCR (MAMA-PCR), were 19 (43.1%) of isolates. In addition, the quinolone resistance gene (qnr) carried by plasmid in Campylobacter isolates was not found in the study. BlaOXA-61 and CmeB (multi-drug efflux pump) genes were detected as 28 (63.6%) and 30 (68.1), respectively. The Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) used for typing the isolates revealed that the band profiles obtained from the isolates were different. In conclusion, this was a very comprehensive study revealing the presence of antibiotic-resistant C. jejuni and C. coli with various virulence genes in patients admitted to a university hospital with acute gastroenteritis. This is of utmost significance for public health. Since campylobacteria are foodborne, zoonotic pathogens and transmission occurs mostly through food. People should have detailed information about the transmission routes of campylobacteria and risky foods. In addition, staff, food processors and caterers, should be trained in hygiene.

Correlation Between Antimicrobial Resistance And Virulence Genes In Klebsiella Pneumoniae Isolates From Egypt.

Objectives: To genotypically assess the relationship between certain resistance and virulence determinants. Method: The cross-sectional study was conducted at Kafrelsheikh University, Egypt, from March 2019 to May 2021, and comprised pathologicalsamples, like blood,sputum, urine, vaginalswabs and wound swabs, that had been taken from patients who had never received treatment. The sample were collected from Kafrelsheikh and Mansoura University hospitals, and Klebsiella pneumoniae isolates were obtained. Resistance and virulence determinants were tested phenotypically. Uniplex polymerase chain reaction was used to evaluate the presence of several resistance accompanied genes and virulence genes in the isolates. Disc diffusion method was used to assess the isolates' susceptibility in accordance with the Clinical and Laboratory Standards Institute criteria for identifying diverse resistance patterns. RESULTS: There were 23 isolatesfrom 16 patients. Of the tested isolates, 22(95.65%)showed drug resistance; 19(82.6%) had multidrug resistance, and 3(13.04%) had extensive drug resistance. There was no case of pan drug resistance. CTX-M-15, NDM, CTX-M-1, VIM-1 and qnr B genes were detected in 14(60.86%), 13(56.5%), 6(26.08%), 6(26.08%) and 6(26.08%) isolates, respectively. Moreover, 6(26.08%) isolates exhibited extended-spectrum ß-lactamase producers, and 12(52.17%) ofsuch isolates contained both CTX-M-1 and CTX-M-15 genes, 6 and 33.3% contained CTX-M-1, CTX M-15 and fox genes. Type 3 fimbriae adhesin mrkD and mucoviscosity regulatory gene uge were found in the tested isolates. However, gene of iron uptake system kfu wasfound in 8(34.78%) isolates, and increased serum survival protein is and mucoviscosity accompanied gene magA were detected in 3(13.04%) isolates. A direct correlation was found among 5 from 8 Klebsiella pneumoniae virulence genes and antimicrobial resistance genes. CONCLUSIONS: There was a direct correlation between the existence of virulence factors and resistance to antimicrobials.

Validation of a random Vibrio parahaemolyticus genomic library by selection of quinolone resistance in a heterologous host.

Vibrio parahaemolyticus is a shellfish-borne pathogen that is a highly prevalent causative agent of inflammatory gastroenteritis in humans. Genomic libraries have proven useful for the identification of novel gene functions in many bacterial species. In this study we prepared a library containing 40 kb fragments of randomly sheared V. parahaemolyticus genomic DNA and introduced this into Escherichia coli HB101 using a commercially available low copy cosmid system. In order to estimate coverage and suitability of the library and potentially identify novel antimicrobial resistance determinants, we screened for the acquisition of resistance to the fluoroquinolone norfloxacin - a phenotype exhibited by V. parahaemolyticus but not the heterologous E. coli host. Upon selection on solid medium containing norfloxacin, 0.52% of the library population was resistant, consistent with the selection of a single resistance locus. End-sequencing identified six distinct insert fragments. All clones displayed fourfold increased norfloxacin MIC compared with E. coli HB101 carrying an empty vector. The common locus contained within resistant clones included qnr, a previously described quinolone resistance gene. These results indicate that the library was unbiased, of sufficient coverage and that heterologous expression was possible. While we hope that this library proves useful for identifying the genetic determinants of complex phenotypes such as those related to virulence, not all norfloxacin resistance genes were detected in our screen. As such, we discuss the benefits and limitations of this approach for identifying the genetic basis of uncharacterized bacterial phenotypes.

Genomic analysis of qnr-harbouring IncX plasmids and their transferability within different hosts under induced stress.

BACKGROUND: Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. METHODS: Complete nucleotide sequences were determined for eight qnr-carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes (n = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. RESULTS: A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. CONCLUSIONS: The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Fluoroquinolone resistance contributing mechanisms and genotypes of ciprofloxacin- unsusceptible Pseudomonas aeruginosa strains in Iran: emergence of isolates carrying qnr/aac(6)-Ib genes.

BACKGROUND: Fluoroquinolones (FQs) including ciprofloxacin (CIP) are key antibiotics for the treatment of Pseudomonas aeruginosa infections, but resistance to FQs is developing as a result of chromosomal mutations or efflux pump effects. Plasmid-mediated quinolone resistance (PMQR) has been recently reported in the Enterobacteriaceae family. This study aimed to investigate the mechanisms of CIP insusceptibility in P. aeruginosa isolates from ICU patients and to characterize their genotypes. METHODS: A total of 40 ciprofloxacin unsusceptible (CIP-US) P. aeruginosa isolates from Tehran hospitals were recruited in this study. A broth microdilution assay was performed to find acquired resistance profiles of the isolates. All isolates were screened for target-site mutations (gyrA and parC), PMQR genes, and efflux pumps (mexB, D, Y, and E) expression. Clonality was determined by random amplified polymorphic DNA (RAPD)-PCR, and genotyping was performed on 5 selected isolates by analyzing 7 loci in the existing multilocus sequence typing scheme. RESULTS: Thirty-eight out of 40 CIP-US isolates (95%) were categorized as MDR. Seven (17.5%) had gyrA mutation in codons 83, and no mutation was detected in parC; 77.5% of the isolates were positive for PMQR genes. Among PMQR genes, qnrB (30%), qnrC (35%), and qnrD (30%) predominated, while qnrA, qnrS, and aac(6)-Ib genes were harbored by 20.5%, 12.5%, and 15% of the isolates respectively. Efflux pump protein expression was observed in 35% of the isolates. After RAPD-PCR, 19 different genotypes were yielded, and 5 of them were classified into sequence types (STs): 773, 1160, 2011, 2386, and 359. CONCLUSION: In this first-time study on P. aeruginosa CIP-US strains from Iranian ICU patients, three main CIP unsusceptibility mechanisms were investigated. A single mutation in one CIP target enzyme could explain high CIP resistance. qnr genes in the isolates can be considered as a CIP-unsusceptibility mechanism among studied isolates. Efflux pumps have more contribution in multidrug resistance than CIP susceptibility. CIP-US isolates of this study have not spread from distinct clonal strains and probably emerged from different sources. STs identified for the first time in this study in Iran should be considered as emerging MDR strains.

The prevalence of plasmid-mediated quinolone resistance genes among Escherichia coli strains isolated from urinary tract infections in southwest Iran.

BACKGROUND: The extensive and inappropriate use of quinolones, which are frequently used as an effective treatment for urinary tract infection (UTI) patients, has led to resistance to these antibiotics. This study was designed to determine the prevalence of quinolones resistance and the presence of plasmid-mediated quinolone resistance (PMQR) genes among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates. METHODS: One hundred and fourteen E. coli isolates were collected from patients' urine samples. The susceptibility of isolates to selected antibiotics was tested by the Kirby-Bauer disk diffusion method. ESBL-producing isolates were identified phenotypically using a combination disk test. Using specific primers, the frequency of aac (6')-Ib, qnrA, qnrB, qnrC, qnrD, qnrS, and qepA genes was investigated by polymerase chain reaction (PCR). RESULTS: Among 26 ESBL-producing isolates, the highest resistance rate was observed toward nalidixic acid (80.8%) and ciprofloxacin (61.5%), respectively. Ninety-seven (85%) of all isolates harbored at least one PMQR gene, the most frequent one being aac(6')-Ib-cr variant (47.4%). Coexistence of aac(6')-Ib-cr variant and qnrB were the most broadly distributed genotype among quinolone resistance isolates. Notably, none of the isolates contained the qnrC, qnrD, and qepA genes. CONCLUSIONS: Our results highlight the significant prevalence of PMQR genes in ESBL-producing E. coli isolates in this region. Also, the aac (6')-Ib-cr variant was the most frequent gene, particularly in ESBL positive isolates. A regular periodic monitoring program is needed to control and hinder the more spread of antibiotic resistance phenomenon and contributed genes among UTI-causing E. coli isolates.

Molecular Characterization of Antibiotic Resistance and Genetic Diversity of Klebsiella pneumoniae Strains.

The aims of this study were the molecular characterization of antibiotic resistance and genotyping of Klebsiella pneumoniae strains isolated from clinical cases in Tehran, Iran. A total of 100 different types of clinical human samples were collected from a major teaching hospital in Tehran, Iran. Bacterial isolates were identified using standard microbiological tests. Antimicrobial susceptibility testing was done according to the latest CLSI guidelines. PCR was used to amplify the gyrA gene in quinolone-resistant isolates and sequencing was performed for the detection of probable mutations between the isolates. The occurrence of plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS) was also investigated by PCR. Finally, genotyping of the strains was performed by PFGE in a standard condition. The susceptibility pattern revealed a high and low level of resistance against meropenem (20%) and trimethoprim (37%), respectively. PCR and sequencing detected mutation in the gyrA gene in 51% of quinolone-resistant K. pneumoniae. According to the susceptibility report, among nalidixic acid-resistant strains, 60.5%, 50%, and 42.9% of isolates contained qnrA, qnrB, and qnrS, respectively. Among ciprofloxacin-resistant strains, qnrA was the most frequent PMQR gene. The PFGE differentiated the strains into 31 different genetic clusters so that the highest number (7/66) was in category A. Our results indicated that the frequency of resistance to various antibiotics particularly trimethoprim, nalidixic acid, and cefoxitin are increasing. The presence of qnr (S and A) genes and point mutation of the gyrA gene were likely to be responsible for the resistance toward nalidixic acid and ciprofloxacin in our strains. Also, the results obtained from genotyping indicated that the K. pneumoniae strains isolated in this study belonged to the diverse clones.