qnrD Genes Research Articles

Exploring Plasmid-Mediated Quinolone Resistance Gene Diversity among Ciprofloxacin-Resistant Proteus Species in a Tertiary Care Hospital of Bangladesh

Background: Increased resistance to fluoroquinolones has been observed in members of the Enterobacteriaceae, including Proteus species. The extensive use of fluoroquinolones and the presence of plasmid-mediated quinolone resistance (PMQR) genes are believed to contribute significantly to fluoroquinolone resistance. This study highlights the prevalence of ciprofloxacin-resistant Proteus species and the presence of specific PMQR genes among them, emphasizing their potential role in quinolone resistance. Method: The study included 30 ciprofloxacin-resistant Proteus isolates collected from wound swab, pus, urine, and blood samples. Identification of Proteus species was performed using culture and biochemical tests. Antibiotic susceptibility test was conducted using the Kirbey-Bauer disc-diffusion method. The minimum inhibitory concentration (MIC) of ciprofloxacin among ciprofloxacin-resistant Proteus species was determined using the agar dilution method. The study aimed to detect specific PMQR genes, namely aac(6´) lb-cr, qnrA, qnrB, qnrD, and qnrS, using polymerase chain reaction (PCR). Result: A total of 42 Proteus species were isolated from 310 various samples. Among the Proteus isolates, 71.43% were ciprofloxacin resistant. The study found that 30% of ciprofloxacin-resistant isolates were positive for aac(6´) lb-cr gene, 13.33% for qnrA and qnrS respectively and 10% for qnrD gene. In total, 60% of ciprofloxacin-resistant Proteus isolates were positive for at least one PMQR gene. The qnrB gene was not detected among ciprofloxacin-resistant Proteus species. The MIC of ciprofloxacin ranged from 8 μg/ml to 128 μg/ml among ciprofloxacin-resistant Proteus isolates. Conclusion: This study suggests aac(6´) lb-cr, qnrA, qnrS, and qnrD genes are emerging in Proteus species, potentially contributing to the development of quinolone resistance and has implications for clinical management and public health. J MEDICINE 2024; 25: 149-154

Antimicrobial susceptibility and virulence gene analysis of Shigella species causing dysentery in Iranian children: Implications for fluroquinolone resistance

Shigella species significantly impact global health due to their role in diarrheal diseases. A 2019–2022 cross-sectional study on 432 stool samples from pediatric patients in Mashhad, Iran, identified Shigella spp. and tested their susceptibility to 12 antimicrobials by the disk diffusion method. The presence of virulence factors, namely ipaH, virA, stx1, and stx2, as well as plasmid-mediated quinolone resistance (PMQR) genes, including qnrA, qnrB, qnrC, qnrD, and qnrS, were ascertained through the utilization of polymerase chain reaction techniques. Sequencing of 15 isolates detected mutations within quinolone resistance-determining regions (QRDRs) at the gyrA and parC genes, indicating fluoroquinolone (FQ) resistance. 19.2 % (83/432) of stool samples contained Shigella, primarily S. sonnei (77.1 %), followed by S. flexneri (21.6 %) and S. boydii (1.2 %). Most isolates were from children under five (55.4 %). All strains had the ipaH gene, lacked stx1 and stx2, and 86.7 % had virA. High resistance was noted for ampicillin and tetracycline (84.3 % each), trimethoprim-sulfamethoxazole (81.9 %), and azithromycin (60.2 %). 87.1 % of isolates were multidrug-resistant (MDR). The most common PMQR genes were qnrA and qnrS (41 % each). The qnrD gene, prevalent in 36.1 % of cases, is reported in Iran for the first time. The most common PMQR profile was qnrADS (15.7 %). Resistance to nalidixic acid and ciprofloxacin was 45.8 % and 12 %, respectively. The Shigella isolates exhibited mutations in the gyrA (at codons 83, 87, and 211) and parC (at codons 80, 84, 93, 126, 128, 129, and 132) genes. The D87Y mutation in the gyrA gene was the most common in Shigella isolates, occurring in 73 % of cases. The F93S and L132T mutations in the parC gene were unique to this study. Empirical FQ therapy in patients infected with MDR Shigella, possessing PMQR determinants and/or mutations in the QRDRs of gyrA and parC, may escalate the risks of secondary diseases, extended treatment duration, therapeutic failure, and resistance spread. Consequently, the necessity for continuous surveillance and genetic testing to detect FQ-resistant Shigella strains is of paramount importance.

Emergence of extensive drug resistance and high prevalence of multidrug resistance among clinical Proteus mirabilis isolates in Egypt

BackgroundProteus mirabilis is an opportunistic pathogen that has been held responsible for numerous nosocomial and community-acquired infections which are difficult to be controlled because of its diverse antimicrobial resistance mechanisms.MethodsAntimicrobial susceptibility patterns of P. mirabilis isolates collected from different clinical sources in Mansoura University Hospitals, Egypt was determined. Moreover, the underlying resistance mechanisms and genetic relatedness between isolates were investigated.ResultsAntimicrobial susceptibility testing indicated elevated levels of resistance to different classes of antimicrobials among the tested P. mirabilis clinical isolates (n = 66). ERIC-PCR showed great diversity among the tested isolates. Six isolates (9.1%) were XDR while all the remaining isolates were MDR. ESBLs and AmpCs were detected in 57.6% and 21.2% of the isolates, respectively, where blaTEM, blaSHV, blaCTX−M, blaCIT−M and blaAmpC were detected. Carbapenemases and MBLs were detected in 10.6 and 9.1% of the isolates, respectively, where blaOXA−48 and blaNDM−1 genes were detected. Quinolone resistant isolates (75.8%) harbored acc(6')-Ib-cr, qnrD, qnrA, and qnrS genes. Resistance to aminoglycosides, trimethoprim-sulfamethoxazole and chloramphenicol exceeded 80%. Fosfomycin was the most active drug against the tested isolates as only 22.7% were resistant. Class I or II integrons were detected in 86.4% of the isolates. Among class I integron positive isolates, four different gene cassette arrays (dfrA17- aadA5, aadB-aadA2, aadA2-lnuF, and dfrA14-arr-3-blaOXA−10-aadA15) and two gene cassettes (dfrA7 and aadA1) were detected. While class II integron positive isolates carried four different gene cassette arrays (dfrA1-sat1-aadA1, estXVr-sat2-aadA1, lnuF- dfrA1-aadA1, and dfrA1-sat2).ConclusionP. Mirabilis ability to acquire resistance determinants via integrons may be held responsible for the elevated rates of antimicrobial resistance and emergence of XDR or even PDR strains limiting the available therapeutic options for management of infections caused by those strains.

Molecular Characterization of Plasmid-mediated Quinolone Resistant Salmonella typhi From Patients Attending Federal Medical Center, Jabi, Abuja, Nigeria

Aims: This study investigates and reports the detection of qnr genes (plasmid-mediated quinolone resistance PMQR) in S.typhi isolated from stool of patients with suspected typhoid fever, in Federal Medical Center, Jabi, Abuja, Nigeria. Study Design: Cross sectional study. Place and Duration of Study: Department of Microbiology, Nasarawa State University, Keffi, between October 2022 and November 2023. Methodology: Salmonella typhi was isolated from stool of patients with suspected typhoid fever using standard culture and microbiological methods. Antibiotic susceptibility testing were performed using the disc diffusion method to investigate the ability of S. typhi to resist some antibiotics. Qnr genes (qnrC,qnrD,qnrS) were detected by PCR and amplification. Results: 17 out of 150 (11.3%) of the samples collected had S.typhi. Antibiotic resistance in the isolates in decreasing order were as follows: cefotaxime (100.0%), amoxicillin/ clavulanic acid (94.1%), nalidixic acid (94.1%), cefuroxime (94.1%), imipenems (88.2%), ceftriaxone/sulbactam (82.4%), ciprofloxacin (82.4%), gentamicin (58.8%), levofloxacin (47.1%) and ofloxacin (29.4%).The commonest antibiotic resistant phenotype was AUG- CTX-IMP-OFX-CN-NA-CXM-CRO-CIP-LBC at 29.4%. Multiple antibiotic resistance (MAR) was observed in 100% (17/17) of the isolates with the common MAR indices being 1.0 (29.4%), 0.7 (23.5%), 0.8 (17.6%) and 0.5 (17.6%). 33.3% of the isolates in FMCJ were positive for qnrC,qnrD,qnrS,qnrA+qnrC and qnrS+ aac(6′)-Ib-cr as all of qnrC qnrD,and qnrS genes were all expressed in the isolates. Conclusion: The S. typhi isolates showed lower resistances to ofloxacin, levofloxacin, and gentamicin, and all isolates were MAR, with resistance to 10 antibiotics being the most predominant. In addition, qnrC,qnrD, and qnrS resistance genes were all expressed in the isolates.

ANALYSIS OF ANTIBIOTIC RESISTANCE GENES OF ESCHERICHIA COLI FROM THE GUT OF THE PIGLETS WITH DIARRHEA

The study is devoted to the analysis of the prevalence of antibiotic resistance genes in cultures of E. coli isolated from the gut of the piglets with diarrhea. With the use of high-throughput sequencing, it was possible to identify 26 antibiotic resistance genes. They belonged to 7 groups of antibiotics: aminoglycosides, beta-lactam antibiotics, quinolones, sulfonamides, tetracyclines, diaminopyrimidine, phenicols. The relative abundance of the QnrD gene was maximal in the studied sample of E. coli isolates (50%). For other sequences of antibiotic resistance genes, the percentage ratio of each separate group of genes did not exceed 10%. The largest number of gene variants was characteristic of the class of genes resistant to beta-lactam antibiotics (AmpC1_Ecoli, OXA-10, OXA-14, OXA-16, Penicillin_Binding_Protein_Ecoli, TEM-143, TEM-166, TEM-215 and TEM-76, TEM- 95).

Molecular characterization of PMQR carrying bacteriophages in effluent discharge samples of Azerbaijan hospitals.

Quinolones are one of the most widely used drugs in medicine. Resistance to this agent has been increased significantly among the nosocomial isolates. The objective of this research was to study generalized transduction, as a potential mechanism for plasmid-mediated quinolone resistance (PMQR) genes acquisition among hospital effluent isolates. Discharge samples from hospital effluent were taken from four medical centers in Azerbaijan. Resident phages were enriched against resident enterobacterial hosts using standard phage enrichment protocols. Polymerase chain reaction (PCR) was used to examine phage stocks and bacterial isolates for the presence of PMQR determinants. All positive bacterial isolates for target genes were subjected to transduction assays. Restriction fragment length polymorphism (RFLP) profiles were determined for cluster analysis. A total of 55 pure phage stocks were prepared from 42 effluents. A total of 95 non-duplicated Gram-negative bacteria were isolated. Thirty-two EcoRV-RFLP profiles were determined for the 40 Escherichia coli phage stocks. Twenty-six of 40 (65%) E. coli phages were positive for qnrB (n = 15), qnrD (n = 7), qnrA (n = 3), and qnrC (n = 2) genes. A total of 34 (35.7%) bacterial isolates were recognized to have any PMQR genes including qnrB (n = 23), qnrD (n = 8), qnrA (n = 5), and qnrC (n = 3) genes. Present research provided a strong evidence for potential role of generalized transduction in persistence and circulation of PMQR genes in health care settings of Azerbaijan.

Prevalence and mechanisms of ciprofloxacin resistance in Escherichia coli isolated from hospitalized patients, healthy carriers, and wastewaters in Iran

BackgroundThis study was aimed to evaluate the prevalence and molecular characteristics of ciprofloxacin resistance among 346 Escherichia coli isolates collected from clinical specimens (n = 82), healthy children (n = 176), municipal wastewater (n = 34), hospital wastewater (n = 33), poultry slaughterhouse wastewater (n = 12) and livestock (n = 9) slaughterhouse wastewater in Iran.MethodsCiprofloxacin minimum inhibitory concentration (MIC) was determined by agar dilution assay. Phylogroups and plasmid-mediated quinolone resistance (PMQR) genes were identified using PCR. Mutations in gyrA, gyrB, parC, and parE genes and amino acid alterations were screened through sequencing assay. The effect of efflux pump inhibitor (PAβN) on ciprofloxacin MICs in ciprofloxacin-resistant isolates was investigated using the microdilution method.ResultsIn total, 28.03% of E. coli isolates were phenotypically resistant to ciprofloxacin. Based on sources of isolation, 64.63%, 51.51%, 33.33%, 14.70%, 10.22% and 8.33% of isolates from clinical specimens, hospital wastewater, livestock wastewater, municipal wastewater, healthy children and poultry wastewater were ciprofloxacin-resistant, respectively. Eighty-one point eighty-one percent (Ser-83 → Leu + Asp-87 → Asn; 78.78% and Ser-83 → Leu only; 3.03% (of ciprofloxacin-resistant E. coli isolates showed missense mutation in GyrA subunit of DNA gyrase, while no amino-acid substitution was noted in the GyrB subunit. DNA sequence analyses of the ParC and ParE subunits of topoisomerase IV exhibited amino-acid changes in 30.30% (Ser-80 → Ile + Glu-84 → Val; 18.18%, Ser-80 → Ile only; 9.10% and Glu-84 → Val only; 3.03%0 (and 15.38% (Ser-458 → Ala) of ciprofloxacin-resistant E. coli isolates, respectively. The PMQR genes, aac(6')-Ib-cr, qnrS, qnrB, oqxA, oqxB, and qepA were detected in 43.29%, 74.22%, 9.27%, 14.43%, 30.92% and 1.03% of ciprofloxacin-resistant isolates, respectively. No isolate was found to be positive for qnrA and qnrD genes. In isolates harboring the OqxA/B efflux pump, the MIC of ciprofloxacin was reduced twofold in the presence of PAβN, as an efflux pump inhibitor. The phylogroups B2 (48.45%) and A (20.65%) were the most predominant groups identified in ciprofloxacin-resistant isolates.ConclusionsThis study proved the high incidence of ciprofloxacin-resistant E. coli isolates in both clinical and non-clinical settings in Iran. Chromosomal gene mutations and PMQR genes were identified in ciprofloxacin resistance among E. coli population.

A qnrD-Plasmid Promotes Biofilm Formation and Class 1 Integron Gene Cassette Rearrangements in Escherichia coli.

Bacteria within biofilms may be exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. Cell-to-cell contact within biofilms facilitates horizontal gene transfers and favors induction of the SOS response. Altogether, it participates in the emergence of antibiotic resistance. Aminoglycosides at sub-MICs can induce the SOS response through NO accumulation in E. coli carrying the small plasmid with the quinolone resistance qnrD gene (pDIJ09-518a). In this study, we show that in E. coli pDIJ09-518a, the SOS response triggered by sub-MICs of aminoglycosides has important consequences, promoting genetic rearrangement in class 1 integrons and biofilm formation. We found that the integrase expression was increased in E. coli carrying pDIJ09-518a in the presence of tobramycin, which was not observed for the WT isogenic strain that did not carry the qnrD-plasmid. Moreover, we showed that biofilm production was significantly increased in E. coli WT/pDIJ09-518a compared to the WT strain. However, such a higher production was decreased when the Hmp-NO detoxification pathway was fully functional by overexpressing Hmp. Our results showing that a qnrD-plasmid can promote biofilm formation in E. coli and potentiate the acquisition and spread of resistance determinants for other antibiotics complicate the attempts to counteract antibiotic resistance and prevention of biofilm development even further. We anticipate that our findings emphasize the complex challenges that will impact the decisions about antibiotic stewardship, and other decisions related to retaining antibiotics as effective drugs and the development of new drugs.

Investigation of plasmid-mediated quinolone resistance genes in carbapenem resistant Enterobacterales

Fluoroquinolones, are effective agents both against gram-positive and gram-negative bacteria. Quinolones show bactericidal effect as a result of inhibition of DNA gyrase and topoisomerase IV enzymes. Main quinolo resistance mechanisms are chromosomal mutations in these enzymes and decreased intracellular accumulation due to efflux pumps or decreased membrane uptake. Recently a new quinolone resistance mechanism mediated by plasmids has been defined. These plasmids carry genes called as qnr. Qnr genes do not cause quinolone resistance but they cause decreased quinolone susceptibility and lead to higher minimum inhibitory concentrations. Currently there are qnrA, qnrB, qnrC, qnrD and qnrS genes. This study was aimed to investigate the presence of plasmid-mediated quinolone resistance determinants in carbapenem resistant Enterobacterales isolates. A total 154 carbapenem resistant Enterobacterales isolates were included in the study. Presence of qnrA, qnrB, qnrC, qnrD and qnrS genes were investigated by multiplex polymerase chain reaction (PCR) method. The results of the PCR amplification revealed that qnrA was detected in two isolates (E6, E85) (1.29%), qnrB was detected in 12 isolates (8.4%) (E32, E43, E46, E61, E62, E84, E94, E149, E 166, E167, E177, E179) and qnrS was detected in six isolates (E15, E25, E57, E63, E70, E80) (4.54%). And one isolate (E9) was both positive for qnrB and qnrS. QnrC and qnrD were not detected in any isolates. Transferable quinolone resistance due to the dissemination of qnr genes may have important impacts in terms of infection control and treatment problems. Survey of plasmid mediated quinolone resistance will help to determine the size of the issue and guide the measures that should be taken to avoid escalation of resistance and dissemination problem.

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

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. 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). 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. 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.

Proteus mirabilis carrying NTEKPC-IId, blaNDM-1, blaOXA-10, aph(3')-VI, qnrD1 and IncQ and Col3M plasmids from a hospital in Recife-PE, Brazil

The present study objective to characterize the clinical aspects of a patient infected with two strains of P. mirabilis and the presence of resistance determinants in the two isolates from a patient at a public hospital in Recife-PE, Brazil. The total DNA of the isolates was extracted and submitted to PCR and amplicon sequencing for the investigation of resistance genes, blaKPC, blaOXA-10, blaOXA-23, blaOXA-48, blaOXA-58, blaVIM, blaIMP, blaSPM, blaGES, blaNDM, qnrD and aac(6')-Ib). Isolate P21-A2 harbored the aac(6')-Ib, blaOXA-10 and qnrD genes. One of the isolates, P20-A2, was selected for plasmid DNA sequencing. The results showed that the patient developed multiple infections with various pathogens including two strains of P. mirabilis. The patient was hospitalized for 103 days, had septic shock of skin, abdominal, pulmonary and ulcer focus, and died. Isolate P20-A2 harbored the genes blaNDM, qnrD, aph(3')-VI, blaKPC and blaOXA-10, and plasmids IncQ and Col3M, together with NTEKPC-IId. To our knowledge, this is the first report of P. mirabilis harboring NTEKPC-IId. Although P. mirabilis is standing out as a cause of nosocomial infections and a resistant multidrug pathogen, this species is still neglected, the emergence of these P. mirabilis isolates harboring aforementioned resistance determinants and the plasmids IncQ and Col3M demonstrate the potential for dissemination of important resistance genes, mainly in the case of P. mirabilis.

Plasmid-Mediated Ampicillin, Quinolone, and Heavy Metal Co-Resistance among ESBL-Producing Isolates from the Yamuna River, New Delhi, India

Antibiotic resistance is one of the major current global health crises. Because of increasing contamination with antimicrobials, pesticides, and heavy metals, the aquatic environment has become a hotspot for emergence, maintenance, and dissemination of antibiotic and heavy metal resistance genes among bacteria. The aim of the present study was to determine the co-resistance to quinolones, ampicillin, and heavy metals among the bacterial isolates harboring extended-spectrum β-lactamases (ESBLs) genes. Among 73 bacterial strains isolated from a highly polluted stretch of the Yamuna River in Delhi, those carrying blaCTX-M, blaTEM, or blaSHV genes were analyzed to detect the genetic determinants of resistance to quinolones, ampicillin, mercury, and arsenic. The plasmid-mediated quinolone resistance (PMQR) gene qnrS was found in 22 isolates; however, the qnrA, B, C, and qnrD genes could not be detected in any of the bacteria. Two variants of CMY, blaCMY-2 and blaCMY-42, were identified among eight and seven strains, respectively. Furthermore, merB, merP, merT, and arsC genes were detected in 40, 40, 44, and 24 bacterial strains, respectively. Co-transfer of different resistance genes was also investigated in a transconjugation experiment. Successful transconjugants had antibiotic and heavy metal resistance genes with similar tolerance toward antibiotics and heavy metals as did their donors. This study indicates that the aquatic environment is a major reservoir of bacteria harboring resistance genes to antibiotics and heavy metals and emphasizes the need to study the genetic basis of resistant microorganisms and their public health implications.

The prevalence and mechanism of fluoroquinolone resistance in Escherichia coli isolated from swine farms in China

BackgroundIt has been demonstrated that swine waste is an important reservoir for resistant genes. Moreover, the bacteria carrying resistant genes and originating from swine feces and wastewater could spread to the external environment. Fluoroquinolones (FQs) are widely used in livestock and poultry for the treatment of bacterial infection. However, resistance to FQs has increased markedly.ResultsIn this study, swine feces and wastewater were sampled from 21 swine farms of seven provinces in China to investigate the prevalence of FQ resistance, including plasmid-mediated fluoroquinolone resistance (PMQR) genes and the occurrence of target mutations. All isolates showed moderate rate of resistance to norfloxacin (43.0%), ciprofloxacin (47.6%), ofloxacin (47.0%) and levofloxacin (38.8%). The percentage of strains resistant to the four FQs antimicrobials was positively correlated with the danofloxacin (DANO) MIC. Among the 74 FQ-resistant isolates, 39 (52.70%) had mutations in gyrA (S83L and D87 to N, Y, G, or H), 21 (28.38%) had mutations in parC (S80I and E84K), 2 (2.70%) had mutations in parE (I355T and L416F), 26 (35.14%) had mutations in marR (D67N and G103S), 1 (1.35%) had mutations in acrR (V29G). While, no mutation was found in gyrB. There were 7 (9.46%) strains carried the qnrS gene, 29 (39.19%) strains carried the oqxAB gene, and 9 (12.16%) strains carried the aac (6′)-Ib-cr gene. In addition, the conjugation assays showed that qnrS, oqxAB and aac (6′)-Ib-cr could be successfully transferred to E. coli J53 from 4 (57.1%), 20 (69.0%) and 5 (55.6%) donor strains, respectively. There were no qnrA, qnrB, qnrC, qnrD and qepA genes detected.ConclusionThe present study showed that DANO-resistant E. coli strains isolated from swine farms had significant cross-resistance to other four FQs antimicrobials. Further study revealed that the resistance mechanisms of swine-derived E. coli to FQs may be attributable to the occurrence of chromosomal mutations (gyrA, parC, parE, marR and acrR genes double-site or single-site mutation) and the presence of PMQR genes (qnrS, oqxAB and aac (6′)-Ib-cr). To the best of our knowledge, one novel mutation marR-D67N was found to be associated with FQ resistance, two mutations parE-L416F and acrR-V29G have never been reported in China.

β-lactam and fluoroquinolone resistance in Enterobacteriaceae from imported and locally-produced chicken in Mozambique.

Plasmid-mediated resistance to β-lactam and fluoroquinolone antibiotics was investigated in Enterobacteriaceae isolated from retailed frozen chickens from Brazil, South Africa and Mozambique. Carcass swabs and the liquid thaw of 33 chickens from each of the three countries constituted the total sample size of 198. Isolates were identified by biochemical tests, antibiotic susceptibility was ascertained by the disc diffusion assay and β-lactamases were detected using the double-disk synergy test. PCR was used to detect the presence of blaCTX-M, blaSHV, blaTEM, blaCMY, blaMOX, blaFOX, blaDHA, qnrB, qnrD, qnrS and qepA genes. A random selection of CTX-M genes was sequenced. The 198 samples yielded 27 (13.6%) putative extended-spectrum β-lactamase (ESBL)-positive isolates, 15 from carcass swabs and 12 from the liquid thaw from 22 chickens with 19, 5 and 3 isolates from South African, Mozambican and Brazilian chicken, respectively. Isolates exhibited the following resistance: ampicillin 100%, ceftriaxone 89%, trimethoprim-sulfamethoxazole 78%, cefotaxime 74%, ciprofloxacin 70%, ceftazidime 67%, cefoxitin 22% and gentamicin 8%. The predominant putative ESBL gene was blaSHV (85%), followed by blaCTX-M (62.9%) and blaTEM (44.4%) whilst blaMOX and blaDHA were the most common pAmpC genes at 33.3%. The predominant plasmid-mediated fluoroquinolone-resistance gene was qepA (22.2%). DNA sequencing identified blaCTX-M-55/-79/-101/-164. ERIC-PCR profiles did not show strong evidence of clonality. The Mozambican population is exposed to a reservoir of plasmid-mediated, and hence mobile β-lactam and quinolone resistance genes via imported, and to a lesser extent, locally produced poultry. This presents a food safety concern.

Study of Plasmid-Mediated Quinolone Resistance in Escherichia coli from Nosocomial Urinary Tract Infections.

The aim of the present study was to study the prevalence of plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac) in Escherichia coli (E. coli) isolated from patients with nosocomial urinary tract infections (UTIs) and its relation to the extended-spectrum β-lactamase (ESBL) production. ; Methods: A cross-sectional study was carried out on 200 non-duplicated isolates of E. coli isolated from patients with nosocomial UTIs. E.coli isolates were subjected to antibiotic susceptibility testing by disc diffusion method, determination of minimum inhibitory concentrations (MICs) of ciprofloxacin by Epsillometer (E) test strips, detection of ESBL production by double disc synergy method and detection of qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac genes by polymerase chain reaction (PCR). ; Results: The antimicrobial susceptibility testing of the isolated E. coli revealed a high frequency of resistance to ampicillin (73.5%), ceftazidime (72%) and imipenem (71.5%). The less frequent resistance was for aztreonam (21.5%), amikacin (36.5%) and gentamicin (38.5%). ESBL production was found in 131 isolates (65.5%) and phenotypic quinolone resistance was detected by MIC in 65 isolates (32.5%), with 52.3% of them showed high resistance to ciprofloxacin with an MIC more than 32 μg/ml. PMQR genes were found in 40 isolates. The frequency of the detected genes was 40%, 37.5%, 35%, 20% and 5% for qnrA, qnrS, qepA, qnrB and oqxA, respectively. Significant association was found between the presence of PMQR genes and ESBL production (P=0.0001). ; Conclusion: The study highlights the prevalence of PMQR genes in E. coli with high association with the ESBL phenotype. This finding is a sign of limited therapeutic options for E. coli.

Potential of industrial composting and anaerobic digestion for the removal of antibiotics, antibiotic resistance genes and heavy metals from chicken manure

Composting and anaerobic digestion techniques are widely used for manure recycling, but these methods have shown conflicting results in the removal of antibiotics, antibiotic resistance genes (ARGs), and heavy metals. In the present study, anaerobically digested chicken manure and various types of composted chicken manure were investigated on an industrial scale. Antibiotics, ARGs, and heavy metals had shown inconsistent results for anaerobic digestion and composting. The different composting processes either declined or completely removed the blaCTX-M, intl1 and oqxB genes. In addition, composting processes decreased the absolute abundance of aac6’-Ib and aadA genes, while increased the absolute abundance of qnrD, sul1, and tet(A) genes. On the other hand, anaerobic digestion of chicken manure increased the absolute abundance of ere(A) and tet(A). High throughput sequencing showed that Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria dominated the total bacterial composition of composted and anaerobically digested samples. Network analysis revealed the co-occurrence of ARGs and intl1. The redundancy analysis showed a significant correlation between some heavy metals and ARGs. Similarly, the bacterial composition showed a positive correlation with the prevalence of ARGs in treated manure. These findings suggest that bacterial community, heavy metals, and mobile genetic elements can play a significant role in the abundance and variation of ARGs during composting and anaerobic digestion. In conclusion, anaerobic digestion and composting methods at industrial scale need to be improved for the effective removal of antibiotics, ARGs and heavy metals from chicken manure.

Quinolone resistance mechanisms among third-generation cephalosporin resistant isolates of Enterobacter spp. in a Bulgarian university hospital.

Background: There have been no reports in Bulgaria about quinolone resistance determinants among Enterobacter spp.Aims: To investigate plasmid and chromosomal quinolone resistance rates among 175 third-generation cephalosporin resistant Enterobacter spp. isolates (167 Enterobacter cloacae complex and eight Enterobacter aerogenes isolates) collected at a university hospital in Varna, Bulgaria, as well as to reveal their association with ESBL/AmpC production and a carriage of specific plasmid replicon types.Methods: PCR, isoelectric focusing, replicon typing, sequencing, and epidemiology typing were carried out.Results: A high level of combined third-generation cephalosporin and quinolone resistant Enterobacter spp. was found − 79.4%. The ESBL production rate was 87%, consisting mainly of CTX-M-15 among E. cloacae complex (in 76%) and CTX-M-3 among E. aerogenes (in 88%). Plasmid mediated quinolone resistance (PMQR) determinants were identified in 57% of the isolates. The most commonly detected PMQR determinants were qnrB (90%), consisting mainly of qnrB1 (in 61%), and qnrB9 (in 27%) of the isolates. Both alleles were transferred with CTX-M-15 genes; transconjugants showed HI2 replicons (for qnrB1 positive transconjugants) and were non-typeable (for qnrB9). One Enterobacter spp. isolate produced qnrB4. QnrA1, qnrS1, and aac(6ʹ)-Ib-cr were detected in single isolates only. QnrC, qnrD, qepA, and oqxAB genes were not found. QnrB was associated with CTX-M-15 production, and qnrS1 was linked to CTX-M-3. Alterations in 83 and 87 positions of gyrB in quinolone-resistance determining regions, and 80 position of parC were detected in high level quinolone resistant isolates. Among all the Enterobacter spp. isolates tested, one predominant clone A was identified (53%).Conclusion: Our data showed the necessity of more prudent use of quinolones and third-generation cephalosporins, because of the risk of promoting dissemination, and selection of multiple resistance determinants (ESBL, PMQR) among Enterobacter spp. isolates in Bulgaria.

Ciprofloxacin Promoted qnrD Expression and Phylogenetic Analysis of qnrD Harboring Plasmids.

Morganella morganii SE10MM harboring quinolone resistance determinant qnrD was investigated in our study. An entirely sequenced novel 2,662 bp qnrD-plasmid pSE10MM was identified and deposited at GenBank under accession number KU160530. Nucleic acid sequence of pSE10MM showed 94-97% similarity to previously detected qnrD-plasmids of Proteus mirabilis strains. Phylogenetic analysis by Geneious 9.0.5 showed clusters of plasmids with possible common origin. Initial expression of qnrD gene was found 12.5 normalized to rpoB housekeeping gene. Subsequently, a sub-minimum inhibitory concentration (1 mg/L) ciprofloxacin exposure resulted in a fold change of 30.06 at 24 hours. In contrast, qnrD-plasmid pSE10MM copy number increased in time from 1.1 to 6.63. Chromosomal mutations of gyrA with S83I, gyrB with S463A, and parC with S80I amino acid substitutions were detected, but no other mutations have occurred as a consequence of ciprofloxacin exposure. Elevated expression of qnrD correlated with that of recA in M. morganii during ciprofloxacin exposure, which indicates SOS-dependent regulation of qnrD. Protective effect of QnrD plays a role in fluoroquinolone-resistant strain even in the presence of chromosomal mutations in gyrase and topoisomerase IV.

Characterization of Quinolone-Resistant Determinants in Tribe Proteeae Isolated from Pet Turtles with High Prevalence of qnrD and Novel gyrB Mutations.

Development of antibiotic resistance in bacteria has challenged significantly in both veterinary and human medicine. In this study, we analyzed the potential risk of pet turtles harboring tribe Proteeae as a source of quinolone-resistant determinants, including plasmid-mediated quinolone resistance (PMQR) genes and target gene alterations in the quinolone resistance-determining region (QRDR). Antimicrobial susceptibility of 54 Proteeae isolates against ciprofloxacin, ofloxacin, levofloxacin, and nalidixic acid was examined. The PMQR genes and QRDR alterations were identified using conventional PCR assays and sequencing. Four isolates were resistant to all quinolones tested in this study. Nine isolates showed resistance to nalidixic acid and showed either intermediate resistance or susceptibility to other tested quinolones. All isolates resistant to one or more tested quinolones harbored mutations in gyrB and some also had gyrA and parC mutations. Of 54, 12 Proteeae isolates displayed the novel E466D, N440T, Q411S, and F417L mutations in gyrB. Among the PMQR genes, 41 (76%) isolates harbored the qnrD gene with the highest prevalence, whereas aac(6')Ib-cr, qnrS, qnrA, and qnrB genes were detected in 28 (52%), 9 (17.0%), 7 (13.0%), and 1 (1.9%) study isolates, respectively. The QRDR analysis of selected mutants revealed that increasing quinolone selective pressure led to a predominance of gyrA mutants. All results indicate that a healthy pet turtle can play as a potential reservoir for quinolone-resistant Proteeae, which it might cause public health risk on pet owners.

QnrD-harboring plasmids in Providencia spp. recovered from food and environmental Brazilian sources

QnrD is a plasmid-mediated quinolone resistance (PMQR) determinant first reported in clinical Salmonella enterica isolates from China, located on nonconjugative plasmids of 4270 bp. Since then, the qnrD gene has been mostly found on plasmids around 2683 bp in Proteus and Morganella genera. However, Providencia spp. strains carrying qnrD-harboring plasmids have only been reported among clinical samples, in France and China. In this paper we describe two plasmids carrying qnrD in Providencia spp. isolated from Brazilian food and coastal waters. These plasmids present high coverage and identity with those recovered in France. Our results emphasize the relevance of the Proteeae tribe as reservoirs of qnrD and include P. rettgeri as a possible environmental carrier of this gene.