Defying the odds: Determinants of the antimicrobial response of Salmonella Typhi and their interplay.

Salmonella Typhi, the invasive serovar of S. enterica subspecies enterica, causes typhoid fever in healthy human hosts. The emergence of antibiotic-resistant strains has consistently challenged the successful treatment of typhoid fever with conventional antibiotics. Antimicrobial resistance (AMR) in Salmonella is acquired either by mutations in the genomic DNA or by acquiring extrachromosomal DNA via horizontal gene transfer. In addition, Salmonella can form a subpopulation of antibiotic persistent (AP) cells that can survive at high concentrations of antibiotics. These have reduced the effectiveness of the first and second lines of antibiotics used to treat Salmonella infection. The recurrent and chronic carriage of S. Typhi in human hosts further complicates the treatment process, as a remarkable shift in the immune response from pro-inflammatory Th1 to anti-inflammatory Th2 is observed. Recent studies have also highlighted the overlap between AP, persistent infection (PI) and AMR. These incidents have revealed several areas of research. In this review, we have put forward a timeline for the evolution of antibiotic resistance in Salmonella and discussed the different mechanisms of the same availed by the pathogen at the genotypic and phenotypic levels. Further, we have presented a detailed discussion on Salmonella antibiotic persistence (AP), PI, the host and bacterial virulence factors that can influence PI, and how both AP and PI can lead to AMR.

Diagnosis of Campylobacter spp. Isolates and Their Antimicrobial Susceptibility Patterns.

This study aimed to reveal antibiotic resistance patterns and molecular characterization of quinolone resistance Campylobacter isolates in patients with diarrhea. Campylobacter spp. isolated from 35.33% of the total samples, most of which were from male patients aged 3 months to 10 years. Identifying isolates at the species level made in MALDI-TOF MS, 82.4% were C. jejuni, and 17.6% were C. coli. Respectively 94% (47/50), 58% (29/50), and 2% (1/50) resistance rates were determined for ciprofloxacin, tetracycline, and erythromycin. While C. jejuni isolates were more resistant to ciprofloxacin and tetracycline than C. coli, they showed no resistance to erythromycin. Quinolone resistance determining region (QRDR) were evaluated by mismatch amplification mutation test and all quinolone resistant strains gave positive results. One of the seven silent mutations identified was specific to this study, and two other novel mutations were also identified. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01199-5.

Multidrug Resistance Profiles and Resistance Mechanisms to ß-Lactams and Fluoroquinolones in Bacterial Isolates from Hospital Wastewater in Bangladesh.

Multidrug resistance (MDR) is one of the deadliest public health concerns of the 21st century, rendering many powerful antibiotics ineffective. The current study provides important insights into the prevalence and mechanisms of antibiotic resistance in hospital wastewater isolates. In this study, we determined the MDR profile of 68 bacterial isolates collected from five different hospitals in Dhaka, Bangladesh. Of them, 48 bacterial isolates were identified as Enterobacteriaceae. Additionally, we investigated the prevalence and distribution of five beta-lactam resistance genes, as well as quinolone resistance mechanisms among the isolates. The results of this study showed that 87% of the wastewater isolates were resistant to at least three different antibiotic classes, as revealed using the disc diffusion method. Resistance to ß-lactams was the most common, with 88.24% of the isolates being resistant, closely followed by macrolides (80.88% resistant). Polymyxin was found to be the most effective against wastewater isolates, with 29.41% resistant isolates. The most common ß-lactam resistance genes found in wastewater isolates were blaTEM (76.09%), blaCTX-M1 (71.74%), and blaNDM (67.39%). Two missense mutations in the quinolone resistance-determining region (QRDR) of gyrA (S83L and D87N) and one in both parC (S80I) and parE (S458A) were identified in all isolates, and one in parE (I529L), which had not previously been identified in Bangladesh. These findings suggest that hospital wastewater acts as an important reservoir of antibiotic-resistant bacteria wherein resistance mechanisms to ß-lactams and fluoroquinolones are obvious. Our data also emphasize the need for establishing a nationwide surveillance system for antibiotic resistance monitoring to ensure that hospitals sanitize their wastewater before disposal, and regulation to ensure hospital wastewater is kept away from community settings.

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

BACKGROUND: This 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. METHODS: Ciprofloxacin 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 (PAßN) on ciprofloxacin MICs in ciprofloxacin-resistant isolates was investigated using the microdilution method. RESULTS: In 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 PAßN, as an efflux pump inhibitor. The phylogroups B2 (48.45%) and A (20.65%) were the most predominant groups identified in ciprofloxacin-resistant isolates. CONCLUSIONS: This 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.

Hotspots sequences of gyrA, gyrB, parC, and parE genes encoded for fluoroquinolones resistance from local Salmonella Typhi strains in Jakarta.

BACKGROUND: Infection of Salmonella enterica subsp. enterica serovar Typhi is the primary etiology of typhoid fever globally and is common in many developing countries, especially those with dense populations and poor environmental sanitation. Antibiotic fluoroquinolones were used for the treatment in the 1980s due to the resistance to the first-line antibiotics. However, many cases of treatment failure of fluoroquinolones in typhoidal patients have been reported from numerous countries in Asia, Europe, Africa, and America. Mutations in quinolone resistance determining regions (QRDR) genes, gyrA, gyrB, parC, and parE, are found in fluoroquinolone-resistant Salmonella Typhi. Contrast reports came from the S. Typhi isolates in Indonesia, mainly Jakarta and the surroundings, obtained from patients with typhoid fever, with good sensitivity to the fluoroquinolones, i.e., nalidixic acid, ciprofloxacin, moxifloxacin, and levofloxacin. The present study, therefore, aimed to identify the hotspot sequences of gyrA, gyrB, parC, and parE genes of the local S. Typhi strains based on their susceptibility to fluoroquinolones from patients with typhoid fever in Jakarta and its satellite cities. RESULTS: A total of 28 isolates were identified as S. Typhi. All isolates were susceptible to nalidixic acid, levofloxacin, and moxifloxacin. Twenty-seven isolates (96.4%) were susceptible to ciprofloxacin, with one isolate (3.6%) being intermediate. The hotspot sequences of gyrA, gyrB, parC, and parE genes from all isolates were identical to the fluoroquinolone-sensitive reference sequence Salmonella enterica subsp. enterica serovar Typhi Ty2 (NCBI GenBank AE014613.1), including the isolate with intermediate susceptibility. The mutation was not found, and amino acid deduced from all hotspots in susceptible and intermediate isolates showed no replacement in all reported codons. CONCLUSIONS: This study showed that the local S. Typhi strains from Jakarta and surroundings were susceptible to fluoroquinolones (nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin), and the hotspot sequences of the gyrA, gyrB, parC, and parE genes were all identical to the reference sequence. Thus, the hotspot sequences of the gyrA, gyrB, parC, and parE genes seemingly were conserved in Jakarta's local S. Typhi strains and could be considered wild type. The phenotypic susceptibility was consistent with the genotypic characteristic without non-synonymous mutations associated with drug resistance.

Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia.

Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::blaCTX-M-2) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.

Pandemic fluoroquinolone resistant Escherichia coli clone ST1193 emerged via simultaneous homologous recombinations in 11 gene loci.

Global growth in antibiotic resistance is a major social problem. A high level of resistance to fluoroquinolones requires the concurrent presence of at least 3 mutations in the target proteins-2 in DNA gyrase (GyrA) and 1 in topoisomerase IV (ParC), which occur in a stepwise manner. In the Escherichia coli chromosome, the gyrA and parC loci are positioned about 1 Mb away from each other. Here we show that the 3 fluoroquinolone resistance mutations are tightly associated genetically in naturally occurring strains. In the latest pandemic uropathogenic and multidrug-resistant E. coli clonal group ST1193, the mutant variants of gyrA and parC were acquired not by a typical gradual, stepwise evolution but all at once. This happened as part of 11 simultaneous homologous recombination events involving 2 phylogenetically distant strains of E. coli, from an uropathogenic clonal complex ST14 and fluoroquinolone-resistant ST10. The gene exchanges swapped regions between 0.5 and 139 Kb in length (183 Kb total) spread along 976 Kb of chromosomal DNA around and between gyrA and parC loci. As a result, all 3 fluoroquinolone resistance mutations in GyrA and ParC have simultaneously appeared in ST1193. Based on molecular clock estimates, this potentially happened as recently as <12 y ago. Thus, naturally occurring homologous recombination events between 2 strains can involve numerous chromosomal gene locations simultaneously, resulting in the transfer of distant but tightly associated genetic mutations and emergence of a both highly pathogenic and antibiotic-resistant strain with a rapid global spread capability.

Epidemic and molecular characterization of fluoroquinolone-resistant Shigella dysenteriae 1 isolates from calves with diarrhea.

BACKGROUND: The widespread distribution of antimicrobial-resistant Shigella has become a recurrent challenge in many parts of the developing world. Previous studies indicate that the host of Shigella has expanded from humans to animals. This study aimed to investigate the prevalence of fluoroquinolone resistance and associated molecular characterization of S. dysenteriae 1 isolated from calves. RESULTS: All 38 unduplicated S. dysenteriae 1 isolates were collected from calves in Gansu Province from October 2014 to December 2016. According to MLST and PFGE analysis, these isolates were separated into 4 and 28 genotypes, respectively. The most common STs identified were ST228 (34.21%, 13/38) and ST229 (39.47%, 15/38), which were first found in the present study. All isolates harbored virulence genes, and the incidence of the seven virulence genes were ipaH (100%), ipaBCD (92.11%), stx (73.68%), ial (57.89%), sen (28.95%), set1A and set1B (0%). According to the results of antimicrobial susceptibilities, 76.32% (29/38) were resistant to fluoroquinolone and showed multidrug resistance. In a study on the polymorphism of quinolone resistance-determining region (QRDR) of gyrA/B and parC/E genes, we identified two mutations in gyrA (Ser83 → Leu and Asp87 → Asn) and parC (Ser80 → Ile and Ser83 → Leu), respectively. Among them, 55.17% (16/29) of resistant strains had the gyrA point mutations (Ser83 → Leu) and parC point mutation (Ser83 → Leu). Moreover, 41.38% (12/29) of isolates had all five point mutations of gyrA and parC. In addition, the prevalence of the plasmid-mediated quinolone resistance (PMQR) determinant genes was also investigated. All 29 fluoroquinolone-resistant isolates were positive for the aac (6')-Ib-cr gene but negative for qepA, except for SD001. In addition, only 6 (20.69%, 6/29) isolates harbored the qnr gene, including two with qnrB (6.90%, 2/29) and four with qnrS (13.79%, 4/29). CONCLUSION: Given the increased common emergence of multidrug resistant isolates, uninterrupted surveillance will be necessary to understand the actual epidemic burden and control this infection.

Distribution of fluoroquinolone resistance determinants in Carbapenem-resistant Klebsiella pneumoniae clinical isolates associated with bloodstream infections in China.

BACKGROUND: The rate of fluoroquinolone (FQ) resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) is high. The present study aimed to investigate the distribution of fluoroquinolone resistance determinants in clinical CRKP isolates associated with bloodstream infections (BSIs). RESULTS: A total of 149 BSI-associated clinical CRKP isolates collected from 11 Chinese teaching hospitals from 2015 to 2018 were investigated for the prevalence of fluoroquinolone resistance determinants, including plasmid-mediated quinolone resistance (PMQR) genes and spontaneous mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes. Among these 149 clinical CRKP isolates, 117 (78.5%) exhibited resistance to ciprofloxacin. The GyrA substitutions (Ser83 → IIe/Phe) and (Asp87 → Gly/Ala) were found among 112 (75.2%) of 149 isolates, while the substitution (Ser80 → IIe) of ParC was found in 111 (74.5%) of the 149 isolates. In total, 70.5% (105/149) of the CRKP isolates had at least two mutations within gyrA as well as a third mutation in parC. No mutations in the QRDRs were found in 31 ciprofloxacin susceptible CRKP isolates. Eighty-nine (56.9%) of 149 were found to carry PMQR genes including qnrS1 (43.0%), aac(6')-Ib-cr (16.1%), qnrB4 (6.0%), qnrB2 (2.7%), and qnrB1 (1.3%). Nine isolates contained two or more PMQR genes, with one carrying four [aac(6')-Ib-cr, qnr-S1, qnrB2, and qnrB4]. The co-existence rate of PMQR determinants and mutations in the QRDRs of gyrA and parC reached 68.5% (61/89). Seventy-four (83.1%, 74/89) PMQR-positive isolates harbored extended-spectrum beta-lactamase (ESBL)-encoding genes. Multilocus sequence typing (MLST) analysis demonstrated that the ST11 was the most prevalent STs in our study. CONCLUSIONS: Mutations in the QRDRs of gyrA and parC were the key factors leading to the high prevalence of fluoroquinolone resistance among BSI-associated CRKP. The co-existence of PMQR genes and mutations in the QRDRs can increase the resistance level of CRKP to fluoroquinolones in clinical settings. ST11 CRKP isolates with identical QRDR substitution patterns were found throughout hospitals in China.

Clinical evaluation of commercial PCR assays for antimicrobal resistance in Mycoplasma genitalium and estimation of resistance-mediated mutation prevalence in Moscow and Moscow region.

Mycoplasma genitalium is a widespread sexually transmitted infection (STI) with growing rate of antimicrobials resistance. In our study, 137 vaginal and 131 urethral M. genitalium-positive swabs were sequentially collected through the work of Reference Center for STI during 2019. For prevalence evaluation of macrolide-resistance mutations three commercially available kits were used: AmpliSens® M. genitalium-ML/FQ-Resist-FL (Central Research Institute of Epidemiology, Russia), ResistancePlus® MG (SpeeDx, Australia), and S-DiaMGRes™ (Diagenode, Belgium). Macrolide resistance mutations were detected in 16% (43 of 268) of samples. Diagnostic characteristics were evaluated against Sanger sequencing. For AmpliSens® M. genitalium-ML/FQ-Resist-FL specificity was shown to be 100% (CI 95%, 98.4-100), and sensitivity was 90.7% (CI 95%, 77.9-97.4). ResistancePlus® MG specificity was 100% (CI 95%, 98.3-100), and sensitivity was 92.1% (CI 95%, 78.6-98.3). S-DiaMGRes™ specificity was shown to be 88.6% (CI 95%, 83.9-92.4), and sensitivity was 100% (CI 95%, 84.4-100). Mutations of parC gene region were detected in 14.5% (38 of 268) using AmpliSens® M. genitalium-ML/FQ-Resist-FL with further validation by Sanger sequencing. Of studied samples, 6.3% (17 of 268) contained both antimicrobials of class resistance mutations. Prevalence of macrolide-resistant M. genitalium in Moscow was 21.7% (23 of 106) and of fluoroquinolone-resistant M. genitaliuim was 20.8% (22 of 106). In Moscow region, macrolide-resistant M. genitalium were 12.3% (20 of 162) and 9.9% (16 of 162) of fluoroquinolone-resistant M. genitalium. All three kits can be used both for epidemiological monitoring of M. genitalium presence and mutation prevalence estimation. In Moscow, macrolide- and fluoroquinolone-resistant mutant prevalence increased in 3.9 and 2.7 times in 3 years.

Bacteriological analysis of Neisseria lactamica isolated from the respiratory tract in Japanese children.

INTRODUCTION: Neisseria lactamica is a commensal bacterium of the upper respiratory tract in humans and is closely related to Neisseria meningitidis. N. lactamica colonization may contribute to preventing N. meningitidis colonization and invasive meningococcal disease. However, the transference of antimicrobial resistance genes from N. lactamica to N. meningitidis has been reported. METHODS: In this study, we aimed to identify N. lactamica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and performed multilocus sequence typing of seven N. lactamica strains isolated from Japanese children. We also analyzed the antimicrobial susceptibility of these strains and the mutations in their antimicrobial resistance genes (penA, gyrA, and parC). RESULTS: All the N. lactamica strains could be identified using MALDI-TOF MS. All strains were of different sequence types (STs), including five new STs. Five strains had intermediate susceptibility, two were resistant to ampicillin, and all had five out of the five known PBP2 mutations. Six strains were resistant to levofloxacin. Among the quinolone-resistant strains, three had GyrA mutations, and three had both ParC and GyrA mutations. CONCLUSIONS: N. lactamica STs may vary in Japanese children, and penicillin- and quinolone-resistant strains may be prevalent. We should pay attention not only to the drug resistance of N. meningitidis but also to the drug susceptibility of N. lactamica whose drug-resistance genes may transfer to N. meningitidis.

Molecular Evaluation of Fluoroquinolone Resistance in Serial Mycobacterium tuberculosis Isolates from Individuals Diagnosed with Multidrug-Resistant Tuberculosis.

Fluoroquinolones (FQ) are crucial components of multidrug-resistant tuberculosis (MDR TB) treatment. Differing levels of resistance are associated with specific mutations within the quinolone-resistance-determining region (QRDR) of gyrA We sequenced the QRDR from serial isolates of MDR TB patients in the Preserving Effective TB Treatment Study (PETTS) with baseline FQ resistance (FQR) or acquired FQ resistance (FQACQR) using an Ion Torrent Personal Genome Machine (PGM) to a depth of 10,000× and reported single nucleotide polymorphisms in ≥1% of reads. FQR isolates harbored 15 distinct alleles with 1.3 (maximum = 6) on average per isolate. Eighteen alleles were identified in FQACQR isolates with an average of 1.6 (maximum = 9) per isolate. Isolates from 78% of FQACQR individuals had mutant alleles identified within 6 months of treatment initiation. Asp94Gly was the predominant allele in the initial FQ-resistant isolates followed by Ala90Val. Seventy-seven percent (36/47) of FQACQR group patients had isolates with FQ resistance alleles prior to changes to the FQ component of their treatment. Unlike the individuals treated initially with other FQs, none of the 21 individuals treated initially with levofloxacin developed genotypic or phenotypic FQ resistance, although country of residence was likely a contributing factor since 69% of these individuals were from a single country. Initial detection of phenotypic resistance and genotypic resistance occurred simultaneously for most; however, phenotypic resistance occurred earlier in isolates harboring mixtures of alleles of very low abundance (<1% of reads), whereas genotypic resistance often occurred earlier for alleles associated with low-level resistance. Understanding factors influencing acquisition and evolution of FQ resistance could reveal strategies for improved treatment success.

Novel mutations in the QRDR region gyrA gene in multidrug-resistance Corynebacterium spp. isolates from intravenous sites.

The resistance to fluoroquinolones in corynebacteria is due to mutations occurring in the quinolone-resistance-determining region (QRDR) of the gyrA gene encoding the enzyme gyrase A subunit. In recent years we can observe an increasing number of infections caused by multidrug-resistant Corynebacterium striatum, Corynebacterium jeikeium and Corynebacterium urealyticum, including wide range of disorders, such as invasive infections. In this study 14 Corynebacterium spp. isolated from intravenous sites were sequenced and new combinations of mutations in the QRDR of the gyrA gene were found in C. jeikeium and C. urealyticum. Nowadays, no study comparing mutations in this region and the susceptibility to fluoroquinolones in C. jeikeium and C. urealyticum has been described. All the isolates that showed double mutation (position 87 and 91) in the QRDR gyrA gene had high MIC to the fluoroquinolones tested.

First report on multidrug-resistant non-encapsulated Streptococcus pneumoniae isolated from a patient with pneumonia.

The non-encapsulated Streptococcus pneumoniae (NESp) has emerged and increased in the clinical setting. The majority of NESp strains have been isolated from the nasopharynxes of healthy carriers and from respiratory specimens of patients with otitis media. NESp strains were shown to be more effective than encapsulated counterparts at forming biofilms. Therefore, NESp should become one of the leading causes of emerging refractory respiratory disease after the introduction of pneumococcal conjugate vaccines. We report the first case of multidrug-resistant - including fluoroquinolone-resistant - NESp isolated from the intrabronchial aspirate of a patient with pneumonia. Drug-resistant NESp infections can possibly emerge as a clinical problem and thus the continuous monitoring of NESp infections is of utmost importance.

gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS: Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS: Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p = 0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p = 0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION: Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.

Conjugal transfer of PMQR from uropathogenic E.coli under high ciprofloxacin selection pressure generates gyrA mutation.

Incidence of high fluoroquinolone resistance has been rising rapidly worldwide. Resistance against fluoroquinolones can be either chromosomal or plasmid mediated. Plasmid mediated quinolone resistant(PMQR) genes impart low level of resistance against fluoroquinolones but provides favorable background for selection of additional chromosomally encoded resistance mechanisms. In the natural habitat, conjugal transfer of PMQR genes provides a vehicle for dissemination of fluoroquinolone resistance. Our study indicated successful transmission of PMQR determinants(aac(6')-Ib-cr, qnrA, qnrB, qnrS, oqxB) from ciprofloxacin resistant clinical uropathogenic E.coli(UPEC) isolates to susceptible E.coliJ53Azide-resistant strain in vitro in presence of high ciprofloxacin (5 µg/ml) selection pressure generating transconjugants that exhibited varied MIC(25-800µg/ml) towards the drug with acquired mutations Ser83Leu and Asp87Asn in the quinolone resistant determining regions(QRDR) in gyrA. Therefore this is a first study of its kind that identified high rate of gyrA mutations among transconjugants selected under high ciprofloxacin pressure resulting from bacterial conjugation a common phenomenon in natural habitat along with PMQR gene transmission which imposes a major public health concern.

New insights into resistance of Helicobacter pylori against third- and fourth-generation fluoroquinolones: A molecular docking study of prevalent GyrA mutations.

BACKGROUND: Fluoroquinolones hinder bacterial DNA replication by inhibiting DNA gyrase. However, mutations, in the QRDR segment of its A subunit (GyrA), cause antibiotic resistance. Here, the interactions of levofloxacin (LVX), gemifloxacin (GXN), and moxifloxacin (MXN) with Helicobacter pylori GyrA, in LVX-resistant vs -sensitive strains, were studied. METHODS: Levoflixacin-sensitive (n = 4) and -resistant (n = 9) H pylori strains, randomly selected from another antibiotic susceptibility study, underwent PCR amplification of gyrA gene, spanning the QRDR segment. The amplified gene fragments were sequenced and aligned. The homology model of H pylori GyrA was built based on that of Escherichia coli, and energy minimization was done. The interaction patterns of LVX, GXN, and MXN with GyrA were analyzed via molecular docking studies. RESULTS: Sequence alignment of the 13 studied strains, created 5 categories of strains: (A) wild type-like (H pylori ATCC26695), (B) N87K-only, (C) D91N-only, (D) N87K + V94L, and (E) D91N + A97V mutations. The minimum inhibitory concentrations (MIC) for LVX-sensitive (category A) and -resistant (categories B-E) strains were <1 mg/L and ≥32 mg/L, respectively. The binding mode of GyrA in category A with LVX identified G35/N87/Y90/D91/V94/G114/S115/I116/D117/G118/D119, as key residues, some residing outside the QRDR segment. Category B strains lost only one interaction (G35), which led to elevated binding free energy (∆G) and full LVX resistance. Categories C-E lost more contacts, with higher ∆G and again full LVX resistance. GXN bound to GyrA of categories A and B via a different set of key residues, while MXN retained the lost contact (G35) in LVX-resistant, category B strains. CONCLUSION: Using molecular docking tools, we identified the key residues responsible for interaction of GyrA with LVX, GXN, and MXN. In the presence of N87K-only mutation, the loss of one of these contacts (ie, G35) led to full LVX resistance. Yet, GXN and MXN overcame this mutation, by retaining all key contacts with GyrA.

Etiology of childhood diarrhoea among under five children and molecular analysis of antibiotic resistance in isolated enteric bacterial pathogens from a tertiary care hospital, Eastern Odisha, India.

BACKGROUND: Although, India has made steady progress in reducing deaths in children younger than 5 years, the proportional mortality accounted by diarrhoeal diseases still remains high. The present hospital based cross sectional study was carried out to understand the prevalence of various bacterial pathogens associated with the diarrhoea cases in under 5 years age group. METHODS: During, 1st September, 2015 to 30th November 2017, all the childhood diarrhoea cases (≤5 yrs) of SCB Medical College in Odisha, India were included in the study. Stool samples were collected and processed for the isolation of causative bacterial pathogen and the isolated bacterial pathogens were subjected to antibiotic sensitivity testing, molecular analysis of drug resistance. Clinical and demographic data were collected and analyzed. RESULTS: Three hundred twenty patients were enrolled in the study during the study period from whom 82 bacterial isolates were obtained indicating a proportional causality of 25.6% for bacterial diarrhoea among children in this region. Entero toxigenic E.coli (ETEC) accounted for majority of the cases and and more than 50% of the strains were found to be multi-drug resistant (resistant to more than 3 class of antibiotics). More than 50% of the strains were resistant to current choice of treatment like ciprofloxacin, ofloxacin and ceftriaxone and 2.4% being resistant to Imipenem. ESBL production was also observed in some of the strains and one isolate harboured the NDM-1 gene. Fluoroquinolone resistance was found to be linked with multiple mutations in the QRDR region followed by PMQR determinants. CONCLUSION: The current study, to the best of our knowledge is first of its kind which demonstrated the etiology of bacterial diarrhoea in children less than 5 years old and identified diarrheogenic E. coli as the predominant enteropathogen in Odisha. Majority of the isolates being multi-drug resistance calls for a continuous surveillance system in the region which will be helpfulin identifying emerging resistance pattern and for developing suitable intervention stategies.

Characterization of Ciprofloxacin Resistance in Laboratory-Derived Mutants of Vibrio parahaemolyticus with qnr Gene.

Ciprofloxacin, a broad-spectrum fluoroquinolone, is a bactericidal antibiotic targeting DNA gyrase and DNA topoisomerase IV encoded by the gyrA and parC genes. Resistance to fluoroquinolones requires the accumulation of multiple mutations including those that alter target genes and increase drug efflux. To examine the development of fluoroquinolones resistance in Vibrio parahaemolyticus, ciprofloxacin induction and selection was used to obtain several resistant V. parahaemolyticus mutants, which showed decreased susceptibilities to quinolones, and increased or decreased susceptibility to other structurally unrelated antibiotics. Quinolone resistance-determining region mutations were characterized, and it was found that gyrA mutations occurred in some of the high-level resistant mutants although qnr was present in both wild-type susceptible and resistant mutant strains. The mutants showed increased qnr expression and exposure to sub-inhibitory concentrations of ciprofloxacin caused a further increase in qnr expression independently of the SOS system. Two mutants demonstrated increased expression of the VmeCD-VpoC pump gene that promotes quinolone efflux. In addition, some of the high-level resistance mutants significantly decreased bacterial fitness. These data suggested that multiple genes contributed to the enhanced ciprofloxacin resistance appeared in V. parahaemolyticus and that acquisition of ciprofloxacin resistance impaired bacterial fitness.

Fluoroquinolone-resistance mechanisms and phylogenetic background of clinical Escherichia coli strains isolated in south-east Poland.

Fluorochinolones are a class of broad-spectrum antimicrobials in the treatment of several infections, including those caused by Escherichia coli. Due to the increasing resistance of bacteria to antimicrobials, an understanding of fluoroquinolone resistance is important for infection control. The aim of this study was to determine susceptibility of clinical E. coli strains to fluoroquinolones and characterize their mechanisms of quinolone resistance. Totally, 79 non-duplicate clinical E. coli isolates included in this study were mainly from skin lesion -36 (45.6%) isolates; 54 (68.4%) isolates were assigned to phylogenetic B2 group. Resistance to ciprofloxacin was found in 20 isolates. In the quinolone resistance-determining region (QRDR) region of gyrA and parC, 4 types of point mutations were detected. Mutations in parC gene were found in all strains with gyrA mutations. Predominance of double mutation in codon 83 and 87 of gyrA (90%) and in codon 80 of parC (90%) was found. Moreover, plasmid-mediated quinolone resistance (PMRQ) determinants (qnrA or qnrB and/or aac(6')-Ib-cr) were present in 5 (25%) out of 20 fluoroquinolone-resistant isolates. Resistance to fluoroquinolones in all of the tested clinical E. coli isolates correlated with point mutations in both gyrA and parC. The majority of fluoroquinolone-resistant strains belonged to D and B2 phylogenetic groups.