Rapid Detection of Quinolone Resistance Mutations in gyrA of Helicobacter pylori by Real-Time PCR

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the Tm values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes.

Characterization of Erysipelothrix rhusiopathiae Isolates from Diseased Pigs in 15 Chinese Provinces from 2012 to 2018

Erysipelothrix rhusiopathiae can cause erysipelas in animals and erysipeloid in humans. Since its recurrence in 2012, swine erysipelas has caused serious losses within the pig industry in China. The aim of this study was to perform multilocus sequence typing and understand the virulence and antimicrobial susceptibility of E. rhusiopathiae isolates in China. Multilocus sequence typing (MLST) of a total of 120 strains was performed, and as a result, three different sequence types were identified, of which ST48 was the main one. Five isolates of each MLST type were randomly selected to be used to challenge mice. ST48 was associated with a higher virulence. Antimicrobial susceptibility was tested using a microdilution technique and, to analyze the resistance mechanism, six strains were selected for genome sequencing. A comparison of the six genomes indicated the presence of a suspected macrolide resistance gene, namely, Erm(A)-like, in erythromycin-resistant strains, which increased the minimum inhibitory concentration (MIC) of erythromycin against E. coli C600 at least four-fold. In addition, three mutations (gyrA86T-I, gyrA90D-N, and parC81S-I) were observed in the quinolone resistance-determining regions (QRDRs) of gyrA and parC in quinolone-resistant strains. After the gyrA gene with the 86T-I mutation or the parC gene with the 81S-I mutation was transfected into E. coli C600, the MIC of enrofloxacin against this strain increased at least two-fold. Our findings provide a theoretical basis for developing antibacterial drugs and may contribute to the clinical prevention and control of E. rhusiopathiae.

Correlation of gyr Mutations with the Minimum Inhibitory Concentrations of Fluoroquinolones among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Bangladesh

Fluoroquinolone (FQ) compounds—moxifloxacin (MOX), levofloxacin (LEV), and ofloxacin (OFL)—are used to treat multidrug-resistant tuberculosis (MDR-TB) globally. In this study, we investigated the correlation of gyr mutations among Mtb isolates with the MICs of MOX, LEV, and OFL in Bangladesh. A total of 50 MDR-TB isolates with gyr mutations, detected by the GenoType MTBDRsl assay, were subjected to drug susceptibility testing to determine the MICs of the FQs. Spoligotyping was performed to correlate the genetic diversity of the gyr mutant isolates with different MIC distributions. Among the 50 isolates, 44 (88%) had mutations in the gyrA gene, one (2%) had a mutation in the gyrB gene, and five (10%) isolates had unidentified mutations. The substitutions in the gyrA region were at A90V (n = 19, 38%), D94G (n = 16, 32%), D94A (n = 4, 8%), D94N/D94Y (n = 4, 8%), and S91P (n = 1, 2%), compared to the gyrB gene at N538D (n = 1.2%). D94G mutations showed the highest MICs for MOX, LEV, and OFL, ranging between 4.0 and 8.0 μg/mL, 4.0 and 16.0 μg/mL, and 16.0 and 32.0 μg/mL, respectively; while the most common substitution of A90V showed the lowest ranges of MICs (1.0–4.0 μg/mL, 2.0–8.0 μg/mL, and 4.0–32.0 μg/mL, respectively). Spoligotyping lineages demonstrated no significant differences regarding the prevalence of different gyr mutations. In conclusion, the substitutions of codon A90V and D94G in the gyr genes were mostly responsible for the FQs’ resistance among Mtb isolates in Bangladesh. Low levels of resistance were associated with the substitutions of A90V, while the D94G substitutions were associated with a high level of resistance to all FQs.

Occurrence of Point Mutations in gyrA and parC Genes of Ciprofloxacin-Resistant Pseudomonas aeruginosa Isolated from Burn Infections

The spread of antibiotic resistant bacteria is a worldwide problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed, through molecular techniques, to detect point mutations in chromosomal genes responsible for the quinolones class of antibiotics resistance. A total of 52 isolates from burn infections were identified using specific primers for P. aeruginosa 16S rDNA. Ciprofloxacin minimum inhibitory concentrations (MIC) were estimated using the agar dilution assay. DNA sequences of the quinolone resistance-determining regions of gyrA and parC were determined for detecting the mutations found in these genes and the relations among the isolates by constructing phylogenetic trees. The results revealed that only 43 (82.7%) of isolates were P. aeruginosa, of which 31 (72.06%) were resistant to different concentrations of ciprofloxacin, ranging between 4 and >32 µg/ml. Twenty six isolates were selected for sequencing, including sensitive, intermediately resistant, and highly resistant to ciprofloxacin. The ciprofloxacin sensitive isolates did not exert any amino acid alterations in gyrA or parC genes; however, a single intermediately resistant isolate had a single mutation at each gene. Of the total resistant isolates (20), 6 isolates had no mutations at different MIC levels, While 14 isolates had Thr-83-Ile substitution in gyrA and Ser-87-Leu substitution in parC, only five isolates had a second mutation, namely Asp-87-Asn, in gyrA. The phylogenetic analysis of the studied groups showed divergence from the P. aeruginosa PAO1 and PAO1OR reference strains due to increased mutations and polymorphisms in studied isolates. In conclusion, P. aeruginosa occurrence was increased in burn infections and the fluoroquinolones in current use are not as effective as before; the main resistance mechanism in local clinical isolates of P. aeruginosa is mutations, where the main target of fluoroquinolones is gyrA gene.

Quinolone-resistant uropathogenic E. coli: is there a relation between qnr genes, gyrA gene target site mutation and biofilm formation?

Introduction. The resistance to quinolone reported in uropathogenic Escherichia coli (UPEC) is commonly caused by mutations in the target site encoding genes such as the gyrA gene. Bacterial plasmids carrying resistance genes such as qnr genes can also transfer resistance. Biofilms produced by UPEC can further aid the development of resistant urinary tract infections (UTIs). Hypothesis. Biofilm production is associated with higher prevalence of quinolones resistance genetic determinants. Aim. To detect the prevalence of qnr genes and gyrA gene mutation among quinolone-resistant UPEC and to investigate the relation between these genetic resistance determinants and biofilm production. Methodology. Catheterized urine samples were collected from 420 patients with evidence of UTIs and processed using standard techniques. Isolated UPEC were screened for quinolone resistance using an antimicrobial susceptibility test. Biofilm production among quinolone-resistant isolates was detected using the tissue culture plate method. All quinolone-resistant isolates were screened for qnr genes (qnrA, qnrB and qnrS) by multiplex PCR and for gyrA gene mutation by PCR-RFLP. Results. Two hundred and sixty-four UPEC isolates were detected from 420 processed urine samples. Out of the identified 264 UPEC, 123 (46.6 %) isolates were found to be quinolone-resistant, showing resistance to 1 or more of the tested quinolones. Of the 123 quinolone-resistant UPEC detected, 71(57.7 %) were biofilm producers. The qnr genes were detected among 62.6 % of the quinolone-resistant UPEC, with an estimated prevalence of 22.8, 32.5 and 37.4 % for qnrA, qnrB and qnrS genes, respectively. Additionally, the gyrA gene mutation was identified among 53.7 % of the quinolone-resistant isolates. We reported a significant association between biofilm production and the presence of qnrA, qnrB and qnrS genes. Furthermore, the gyrA gene mutation was significantly associated with biofilm-producing isolates. The coexistence of qnr genes, gyrA gene mutation and biofilm production was demonstrated in almost 40 % of the quinolone-resistant isolates. Conclusions. A significantly higher prevalence of qnr genes (qnrA, qnrB and qnrS) as well as the gyrA gene mutation was found among biofilm-forming UPEC. The reported coexistence of these different resistance mechanisms could aggravate quinolone resistance. Therefore, monitoring of resistance mechanisms and a proper stewardship programme are necessary.

Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa

Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of blaOXA-51. Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were blaOXA-51-positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

First report of Klebsiella aerogenes Inciting Stem Rot of Pearl Millet in Haryana, India

Pearl millet [Pennisetum glaucum (L.) R. Br. Syn. Pennisetum americanum (L.) Leeke] is the oldest and widely cultivated millet in Asian and African countries, mostly grown over low fertile soils in more than 40 countries covering an area of 312.00 lakh hectares (FAOSTAT 2017). In Haryana, crop was grown over an area of 4.30 lakh hectares during Kharif 2019. Pearl millet is prone to many fungal and bacterial diseases. During 2018 to 2020, a new devastating diseas exhibiting stem rot like symptoms was observed in pearl millet growing regions in Indian state of Haryana. The isolated disease causing agent was a bacterium, where 16S rDNA-based nucleotide sequence deposited in NCBI GenBank (Accession nos. MZ433194.1) conferred its nearness to Klebsiella aerogenes (Hormaeche and Edwards 1960) Tindall et al. 2017. Further, DNA gyrase genomic sequence (NCBI Accession nos. MZ707528.1) also stayed its high homology to K. aerogenes. Klebsiella usually known to cause diseases in humans and animals, and also has been found inciting different kind of rots in different plantations viz. top rot in maize (Huang Min et al. 2016). Pearl millet is susceptible to minor bacterial diseases viz. bacterial leaf streak (Xanthomonas campestris), bacterial leaf spot (Pseudomonas syringae) and leaf stripe (P. avenae). Earlier, among the plant pathogenic bacterial entirety, only Erwinia chrysanthemi is known to cause stem rot diseases in sorghum (Saxena et al. 1991) amongst different types of millet. Extensive disease survey of pearl millet growing regions (Hisar, Bhiwani, Rewari, Mohindergarh and Bawal districts of Haryana having an altitude of 215, 225, 245, 262 and 266 m, respectively) in rainy seasons of 2019 and 2020 revealed the prevalence of typical stem rot disease, representing up to 70% disease incidence in the infected fields. The pieces of symptomatic stem of different plants were collected from two locations (Hisar and Bhiwani) and associated organism was isolated following the techniques of Janse (2005). The resulting growth of bacterial cultures were further purified on nutrient agar (NA) media using streak plate technique where colony growth of both the isolates were observed as morphotypes. The resulting bacteria were gram-negative and rod-shaped. Colonies were round and creamish white on NA. Isolated morphotypes were positive for indole production, methyl red, Voges Proskauer’s test, citrate utilization, arabinose, mannitol, rhamnose and sucrose, whereas negative for glucose, adonitol, lactose and sorbitol tests. Biochemical tests were performed following standard methods (Holt et al. 1994). Molecular analysis of both isolates was performed using two sets of primers (universal 16S rRNA gene and genus-specific gyrA gene). The gyrA fragment (F: 5ʹ-CGCGTACTATACGCCATGAACGTA-3ʹ; R: 5ʹ-ACCGTTGATCACTTCGGTCAGG-3ʹ) has been adopted as Klebsiella genus-specific gene (Brisse and Verhoef 2001). The quality and quantity of the isolated genomic DNA were analyzed using NanoDrop-2000 (Thermo Fisher Scientific, USA) and resolved in 1% (w/v) agarose gel. Thereafter, visualized in gel documentation to confirm a single band of high-molecular-weight DNA. The fragment 16S rDNA was amplified using 27F and 1492R primers, where a single discrete PCR amplicon of 1500 bp was observed in 1% (w/v) agarose gel. Similarly, the gyrA gene was amplified using 09510F and 09510R primers that conferred a single discrete band of 400 bp. The forward and reverse DNA sequencing reaction of purified PCR amplicons (16S rDNA and gyrA) was carried out using BDT v3.1 Cycle sequencing kit on a genetic analyzer to generate gene sequences. The consensus sequences of both gene were generated from forward and reverse sequences data using aligner software. The obtained sequences of both genes were compared with the available nucleotide sequences in the NCBI using the blast 2.2.9 system (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch). The sequenced PCR amplicons showed up to 100% similarity with Klebsiella aerogenes 16s RNA nucleotide sequences (Accession nos. NR102493.2, MT373521.1; MF682950.1; MF462979.1 etc.). The bacterium also showed high nucleotide homology to K. aerogenes gyrA gene sequences (Accession nos. LR607333.1; CP035466.1; CP049600.1 etc.). The molecular phylogenetic analysis was done by the maximum likelihood method based on the Tamura-Nei model, and 1000 replicates for bootstrap testing in MEGA 7.0 software. The analysis involved 16 nucleotide sequences and evolutionary distances were computed. The 16s RNA based phylogenetic tree raised using MEGA7 (Kumar et al. 2016) elucidates that Klebsiella aerogenes Hisar formed a cluster with three K. aerogenes strains (Accession nos. MZ577128.1, MT373521.1 and MT 373520.1), whereas K. aerogenes Bhiwani displayed higher homology to NCBI sequences viz. MF682950.1, MT355368.1, MW331687.1and LC515412.1. Bacterial suspension was prepared by suspending bacterial cells into sterile water and cell density was adjusted to 1×107 colony forming unit/ml. For pathogenicity, leaf whorl inoculation (10 ml suspension/ whorl) was done on 15 days old seedlings of pearl millet genotype 7042S raised under controlled conditions (Temperature 35±2°C and more than 80% Relative Humidity). The pathogenicity was proved under field conditions as well. Initial symptoms were observed 4-5 days after inoculation as long streaks on leaves. Soon a spike in number of these leaf streaks was observed. Thereafter, water-soaked lesions appeared on the stem at 20-25 days after inoculation which later on turned brown to black. Severely diseased plants were dead, exhibiting hollowing of the stem and drying of leaves. The infected stem pith disintegrated and showed slimy rot symptoms and the pearl millet clumps toppled down. The rotten stems of both inoculations were again cut in to small pieces and the reisolated bacterium showed exactly the same morphological, biochemical and molecular characteristics. To our knowledge, this is the first report of stem rot of pearl millet incited by K. aerogenes in south-western regions of Haryana, India. Because the stem rot caused by K. aerogenes poses a significant threat to pearl millet cultivation, further research on biology, epidemiology and management choices is needed.

Phenotypic and Molecular Patterns of Resistance among Campylobacter coli and Campylobacter jejuni Isolates, from Pig Farms

The purpose of this research was to characterize the antibiotic resistance patterns of Campylobacter spp. isolated from commercial farrow to finish farms in Greece, and analyze the relevant molecular resistance mechanisms among the resistant Campylobacter isolates. Susceptibility testing to five different classes of antibiotics was performed in 100 C. coli and 100 C. jejuni, previously isolated and identified. All isolates were found susceptible to meropenem. Very high rates of resistance were recorded for tetracyclines (84.5%), medium rates of resistance were recorded regarding quinolones (23%), and low and very low rates of resistance were identified for macrolides such as erythromycin and aminoglycosides (12% and 4%, respectively). Only 12.5% of the Campylobacter isolates displayed MDR. Regarding the molecular mechanisms of resistance, all ciprofloxacin resistant isolates hosted the mutant type Thr-86-Ile region of the quinolone resistance-determining region (QRDR) of the gyrA gene. In all erythromycin resistant isolates, the transitional mutations A2075G and A2074C in the 23S rRNA gene were only amplified. Molecular screening of tetracycline resistance genes indicated that the vast majority of Campylobacter isolates (92.3%) were positive for the tet(O) gene. In summary, these findings and especially the very high and medium rates of resistance for tetracyclines and fluroquinolones, respectively recommend that a continuous monitoring of Campylobacter isolates susceptibility in combination with the proper use of antimicrobials in livestock production is of great importance for public health.

First Report of Enterobacter cloacae Causing Bulb Decay on Garlic in China

Enterobacter cloacae is a symbiotic bacterium, which is one of the species in intestinal microbiota in many humans and animals. In some cases, it causes harmful diseases in humans. More and more studies showed that E. cloacae caused disease on plants, such as macadamia, ginger, mulberry, onion, chili pepper and rice. Garlic (Allium sativum L.) is one of crops with economic importance in the world. It is also widely grown in China. During 2018 to 2020, the naturally infected garlic bulbs from garlic fields in Kaifeng of Henan Province (34.55° N; 114.78° E) showed dry brown discoloration and rot symptoms. The diseased garlic seriously affected its edible value. Voucher specimens collected on June, 2019 were deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU190620. To identify the causal agent of this disease, the bulb tissues of infected garlic were surface-disinfested in 0.6% sodium hypochlorite, dipped in75% ethanol, and then dipped in sterile distilled water. These bulbs were plated on LB medium and incubated at 37℃. A number of white colonies grew on the medium after plating for 16 h. All colonies were round, white, opaque, smooth, and gram-negative, which is a typical characteristic of Enterobacter. To confirm the initial identification of the isolated bacterium, the fragments of 16S rRNA gene and gyrA gene of 6 colonies were amplified, respectively. The PCR products were purified and sequenced. All 16S rRNA and gyrA sequences were identical to each other. The sequences of 16S rRNA gene and gyrA gene were deposited in GenBank with accession numbers MW730711 and MW768876, respectively. BLAST searches were conducted using the sequences of 16S rRNA and gyrA. The results showed 99.72%, and 96.91% identity with the corresponding sequences of E. cloacae strain CBG15936 (CP046116.1), respectively. Phylogenetic trees were performed using the neighbor-joining (NJ) method of MAGA X based on the sequences of 16S rRNA gene and gyrA gene. Phylogenetic tree indicated that isolates are most likely E. cloacae. Pathogenicity tests were performed by puncturing garlic bulbs with a hypodermic needle, followed by dipping in bacterial suspension with the concentration of 2×108 CFU for 5 minutes. As control, the garlic bulbs were treated with sterile water. The inoculated and control were incubated at 30°C. 7 days after inoculation, brown discoloration and rot were developed on all inoculated garlic bulbs. No symptoms were observed in the control group.The symptoms were similar to that observed on the original diseased garlic bulbs. The garlic bulbs in inoculated and control were ten replicates in each independent biological experiments. The pathogenicity tests were conducted three times with similar results. The bacteria were re-isolated from the symptomatic diseased garlics and confirmed as E. cloacae by morphological and sequence analyses as above. The re-isolated bacteria were identified by biochemical and physiological characteristics using API 20E strips. The results of the identification were identical to those of the edible ginger strains and the chili pepper strains. As far as we know, this is the first report of bulb decay on garlic caused by E. cloacae. The results are of great significance not only for the management of garlic bulbs during postharvest handling and storage, but also for the further research of opportunistic human pathogens E. cloacae.

Retrospective analysis of infection and antimicrobial resistance patterns of Mycoplasma genitalium among pregnant women in the southwestern USA

BackgroundMycoplasma genitalium is a sexually transmitted infection (STI) pathogen. There have been no published studies concerning symptomatology, prevalence data, antibiotic resistance profiling or reports of co-infection with other STI in pregnant women.ObjectiveTo describe these characteristics among pregnant women attending prenatal clinics in a large tertiary care centre.DesignRemnant genital samples collected from pregnant women between August 2018 and November 2019 were tested for M. genitalium and Trichomonas vaginalis by the transcription-mediated amplification technique. Specimens with detectable M. genitalium RNA were sequenced for 23S rRNA mutations associated with azithromycin resistance and parC and gyrA mutations associated with resistance to moxifloxacin. Demographic, obstetric and STI co-infection data were recorded.ResultsOf the 719 samples, 41 (5.7 %) were positive for M. genitalium. M. genitalium infection was associated with black race, Hispanic ethnicity and young age (p=0.003, p=0.008 and p=0.004, respectively). M. genitalium infection was also associated with T. vaginalis co-infection and Streptococcus agalactiae (group B Streptococcus) colonisation (p≤0.001 and p=0.002, respectively). Of the 41 positive samples, 26 (63.4%) underwent successful sequencing. Eight (30.8%) had 23S rRNA mutations related to azithromycin resistance. One of 26 (3.8%) positive samples with sequencing results had the gyrA gene mutation and 1 of 18 sequenced samples (5.6%) had the parC gene mutation associated with moxifloxacin resistance.ConclusionsPrevalence rates of M. genitalium in pregnant women was 5.7%. M. genitalium infection disproportionately affects young black women co-infected with T. vaginalis. Pregnant women remain at risk for persistent infection with M. genitalium due to decreased azithromycin susceptibility.