Nalidixic Acid Resistance Research Articles

Antimicrobial resistance, virulence, and molecular characterization of aquatic, clinical, and environmental Vibrio parahaemolyticus isolated from Ningbo, China

Abstract Vibrio parahaemolyticus is the leading cause of illness associated with seafood consumption globally. In this study, we examined 136 V. parahaemolyticus isolates from Ningbo, China for antimicrobial resistance phenotypes, genotypes, virulence factor genes (tdh, trh, T3SS) and molecular typing. Carbenicillin resistance (100.0%) was the most prevalent in all isolates followed by ampicillin with 46.1% in aquatic, 9.2% in clinical, and 83.3% in environmental isolates. Trimethoprim‐sulfamethoxazole (7.7%), streptomycin (3.1%), tetracycline (3.1%), and nalidixic acid (1.6%) resistance were only present in aquatic isolates. The virulence factor genes tdh (83.1%) and trh (10.8%) were found in clinical isolates while tdh was carried by aquatic (6.2%) and environmental (83.3%) isolates. T3SS2α genes were present in tdh +/trh− isolates and T3SS2β genes were present only in trh + isolates. Molecular typing by enterobacterial repetitive intergenic consensus‐polymerase chain reaction classified our 136 isolates into nine clusters at a 0.66 similarity and aquatic isolates showed high degrees of genetic diversity. Our findings characterized the risk factors among aquatic, clinical, and environmental V. parahaemolyticus isolates from Ningbo, China.Practical ApplicationsThe threat of pathogenic Vibrio parahaemolyticus to seafood safety is more serious than ever before as antimicrobial resistance has emerged and developed globally in the past decades. This study emphasizes the antimicrobial susceptibility, virulence, and molecular characterization of aquatic, clinical, and environmental V. parahaemolyticus isolated in Ningbo, which will be helpful for improving seafood safety management.

Antibiotic sensitivity pattern in culture positive typhoid fever cases isolated at Patan hospital

Background: Enteric fever is one of the most common systemic infections of developing countries like Nepal. The changing trend of antibiotic susceptibility and increasing rate of resistance to the commonly used antibiotics has been of great concern in the proper treatment and prevention of this disease. This study aims to investigate the current antibiotic susceptibility pattern of Salmonella typhi and paratyphi A and B.
 Materials and Methods: This study was carried out at Patan hospital of Lalitpur, Nepal from ____ to -------. Blood culture sample were collected from suspected patients presenting to different departments of the hospital and tested microbiologically by standard procedure. Antibiotic susceptibility test was performed by Kirby Bauer Disc Diffusion method and results were interpreted by National Committee for Clinical Laboratory (NCCLS) guidelines.
 Results: Out of the total sample of 212, 40(18.8%) cases were isolated as Salmonella species. Out of these, 29(72.5%) cases were S typhi and 11(27.5%) were S paratyphi. Isolates were mainly from adult age group. Regarding the antibiotic susceptibility pattern, the sensitivity to chloramphenicol was 95% and to cotrimoxazole 97% whereas the sensitivity to ciprofloxacin was 52.5% and 27.5% for Ofloxacin. Nalidixic Acid Resistance Salmonella typhi strain was 60.0 percent. Sensitivity to ceftriaxone, meropenem and colistin was found to be 100 percent.
 Conclusions: A high degree of variability is seen in antimicrobial sensitivity pattern with very high degree of sensitivity to the historically used antibiotics like chloramphenicol and cotrimoxazole. Unfortunately, sensitivity to quinolones was seen to be very low, which were used widely in the last two decades.

Virulent Escherichia coli strains among Egyptian patients with acute diarrhoea versus urinary tract infection, and their antibiotic susceptibility

Background and study aimsDiarrhoea and urinary tract infection (UTI) are common clinical problems. Meanwhile, Escherichia coli (E. coli), is the commonest bacterial pathogen reported in both of them. This study aimed to evaluate the pathogenic E. coli (PEC) in stool of acute diarrhoea and urine of UTI regarding their virulence genes and their influence on the susceptibility to routinely prescribed antibiotics. Patients and methodsTwenty two stool and another 22 urine samples of patients with acute diarrhoea and UTI respectively were collected from patients admitted at Kasr Al-Ainy Hospital, Faculty of Medicine, Cairo University, Egypt. E. coli isolation, identification of their phyla; chuA, yjaA, and TspE4.C2, and further identification of 10 virulent genes; fimH, papC, papG//, papG///, papEF, afa, sfa, CNF1, iroN & hlyA was performed. Antibiotic susceptibility was studied against quinolones, gentamicin (GM), and trimethoprim-sulphamethoxazole (TMP-SMX). ResultsThe studied virulence genes were comparably detected in both pathogenic samples. In diarrheogenic E. coli (DEC); phylum A was significantly related to both ciprofloxacin (CIP) and TMP-SMX resistance, and both of the virulence genes fimH and iroN were significantly related to all the studied antibiotics resistance, while afa was significantly related to nalidixic acid (NA) resistance. In uropathogenic E. coli (UEC); phylum D was significantly related to CIP and levofloxacin resistance, and both of the virulence genes fimH and iroN were significantly related to most of the studied antibiotics resistance. ConclusionThe isolated PEC was evidently and broadly resistant to the studied antibiotics, with limited influence of their phyla and virulence genes (fimH and iroN).

Phenotypic and Genotypic Examination of Antimicrobial Resistance in Thermophilic Campylobacter Species Isolated from Poultry in Turkey.

IntroductionThe study aimed to isolate thermophilic Campylobacter from chickens raised three rearing methods, determine its antimicrobial susceptibilities, and examine resistance-related genes by PCR.Material and MethodsCloacal swabs or intestinal contents were taken in Istanbul, Sakarya, and Izmir provinces. Chickens were from small village-based family-run businesses (n = 70), organically raised (n = 71), and conventionally raised broilers (n = 79). The samples were cultured on modified charcoal cefoperazone desoxycholate (mCCD) agar. Suspect isolates were identified with multiplex PCR (mPCR). As per EUCAST standards, MIC values were derived by broth microdilution for tetracycline, ciprofloxacin, nalidixic acid, kanamycin, gentamicin, and erythromycin in isolates of C. jejuni (n = 98) and C. coli (n = 83).ResultsIn C. jejuni, 78.6% tetracycline, 87.8% ciprofloxacin, and 81.6% nalidixic acid resistance was detected, but none was to kanamycin, gentamicin, or erythromycin. In C. coli, 98.8% ciprofloxacin and 63.9% nalidixic acid resistance was detected, whereas resistance to non-quinolones was not observed. C257T (Thr-86-Ile) mutation in the gyrA gene of all phenotypically quinolone-resistant isolates was detected through a mismatch amplification mutation assay PCR (MAMA-PCR). It emerged that all isolates bore the tet (O) resistance gene.ConclusionCommon tetracycline, nalidixic acid, and ciprofloxacin resistance exists in Campylobacter isolated from chickens raised three rearing methods.

High frequency of mutations in gyrA gene associated with quinolones resistance in uropathogenic Escherichia coli isolates from the north of Iran.

Objective(s):Regarding the global burden of uropathogenic Escherichia coli (UPEC) infections, prevention and treatment of such infections play a significant role in healthcare management. The inordinate use of fluoroquinolones led to a worldwide spread of quinolone-resistant strains. Therefore, this study aimed to investigate mutations in codons 83 and 106 of gyrA gene in UPEC isolates in the north of Iran.Materials and Methods:This cross-sectional study performed on a total of 223 UPEC isolates which were recovered within 6 months in 2017. Isolates were identified and confirmed by standard microbiologic tests, and antimicrobial susceptibility testing was carried out by disk diffusion and E-test methods. PCR reaction was performed to amplify gyrA gene, and PCR-RFLP was performed using BsiEI and BstUI restriction enzymes to investigate mutations in gyrA gene.Results:The nalidixic acid, ciprofloxacin, ofloxacin, and norfloxacin resistance rates were 61.9%, 50.2%, 48.25, and 45.3%, respectively. Overall, 55.2% of E. coli isolates had a mutation in gyrA gene in codon 83, and 20.2% in codon 106. Also, 15.2% of isolates had simultaneously mutation. Moreover, a significant association was found between mutations in gyrA gene and quinolone and fluoroquinolones resistance pattern of UPEC isolates.Conclusion:Our results revealed a high level of quinolone resistance associated with the mutations in gyrA among the clinical isolates of UPEC in our region. To the best of our knowledge, this study is the first investigation on the role of gyrA alteration in quinolone resistance among UPEC isolates from the north of Iran.

Occurrence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from domestic animals from Southern Ecuador

ABSTRACT: Occurrence and antimicrobial resistance profiles of Campylobacter jejuni and C. coli strains isolated from fecal samples of dogs, pigs, cows and hens in southern Ecuador were studied. Of the 250 samples studied, 84 (33.6%) were Campylobacter positive, with C. jejuni being more frequent (78.6%) than C. coli (21.4%), with the exception of porcine samples, from which C. coli was the only species isolated. Multidrug resistance was reported in 10 Campylobacter strains (11.9%), four C. jejuni and six C. coli, and was always associated with nalidixic acid and ciprofloxacin resistance. All of the amoxicillin-resistant strains were susceptible to amoxicillin-clavulanic acid and were therefore beta-lactamase producers. However, one strain of C. jejuni remained resistant with additional resistance to gentamycin. This is an uncommon resistance pattern in Campylobacter and could reflect different resistance mechanisms.

Molecular Characterization of Salmonella enterica Serovar Enteritidis, Genetic Basis of Antimicrobial Drug Resistance and Plasmid Diversity in Ampicillin-Resistant Isolates.

Salmonella enterica serovar Enteritidis is the most common cause of human salmonellosis worldwide. In this study, all clinical isolates of Salmonella Enteritidis recovered between January 2008 and June 2014 in a Spanish region (491) were screened for antimicrobial drug resistance and the phage type (PT) was determined for a significant number (265). PT1, PT14b, PT56, PT6, PT4, and PT8 were the predominant PTs, accounting together for 82% of the isolates. A total of 38.3% of the isolates were susceptible to all antimicrobials tested, 46.4% and 6.1% isolates were resistant to nalidixic acid and ampicillin, respectively, and single isolates were resistant to two (ampicillin and nalidixic acid) or six (ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim) agents. Nalidixic acid resistance was statistically associated with PT1 and PT14b (p < 0.05, 95% CI), and ampicillin resistance with PT6/PT6a (p < 0.05, 95% CI). All ampicillin-resistant isolates (30) carried a plasmid-encoded blaTEM-1. All except one harbored the virulence plasmid specific of Salmonella Enteritidis (IncFIIA + IncFIB; 28 isolates) or a blaTEM-1-positive variant herein (IncFIIA + IncFIB; 1 isolate). Five additional blaTEM-1 plasmids, of the ColE1, IncX, IncF, and IncI incompatibility groups, were identified. The IncI plasmid, found in the single multidrug-resistant isolate, carried the strAB and sul2 genes together with genes of the virulence plasmid, including the spv operon. The obtained results highlight the high diversity of blaTEM-1 plasmids conferring ampicillin resistance in Salmonella Enteritidis, and support clonal expansion as the main cause of nalidixic acid resistance in this serovar.

Fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli from poultry and human samples assessed by PCR-restriction fragment length polymorphism assay.

The objective of this study was to determine fluoroquinolone resistance in Campylobacter spp from poultry and human isolates. Forty-one Campylobacter jejuni isolates (30 of poultry origin and 11 of human origin) and 11 Campylobacter coli isolates (10 of human origin and 1 of poultry origin) were examined for ciprofloxacin, norfloxacin, and nalidixic acid resistance using the minimal inhibitory concentration (MIC) method. Thereafter, the isolates were analyzed by PCR–Restriction Fragment Length Polymorphism (RFLP) assay for detection of Thr-86 mutation. Finally, DNA sequencing was performed for confirmation of gyrA gene mutation. A complete correlation was observed between MICs, PCR-RFLP assay, and sequencing. The results revealed high quinolone resistance rates for C. jejuni (100%) and C. coli (100%) isolates obtained from poultry and moderate resistance for C. jejuni (9.1%) and C. coli (40%) samples of human origin. A mutation in codon 86 of the gyrA gene with a Thr-to-Ile substitution is reported to be the main cause of high resistance to quinolones. This mutation can be analyzed by PCR-RFLP assay, which has been proven to be a simple and fast method for the detection of fluoroquinolone resistance in Campylobacter spp.

Frozen White-Leg Shrimp (Litopenaeus vannamei) in Korean Markets as a Source of Aeromonas spp. Harboring Antibiotic and Heavy Metal Resistance Genes.

As the most consumed shrimp variety, white-leg shrimp (Litopenaeus vannamei) owns a high market demand in Korea. This study sought to screen the frozen white-leg shrimp for Aeromonas spp. harboring antimicrobial and heavy metal resistance characteristics. A total of 44 Aeromonas spp. strains were isolated and tested for antibiotic susceptibility and heavy metal tolerance followed by PCR-based detection of resistance genes and integrons. It was observed that resistance to ampicillin and oxacillin was 100% among isolates. Besides, 95%, 89%, 86%, 80%, 66%, and 43% of the isolates were resistant to nalidixic acid, tetracycline, cephalothin, streptomycin, trimethoprim-sulfamethoxazole, and imipenem, respectively, and less resistance to other antibiotics was also observed. Cr resistance was the highest (91%) among five heavy metals tested, whereas 57%, 32%, 20%, and 9% of the isolates were tolerant to Cu, Pb, Cd, and Hg, respectively. The PCR assays implied the presence of qnrB, qnrS, tetA, tetE, aac(6')-Ib, and aphAI-IAB, and intI1 genes among 80%, 77%, 18%, 30%, 9%, 0.25%, and 82% of the isolates, respectively. There were 35 (80%) integron 1-positive isolates harboring qacE2, dfrA1, orfC, orfD, aadB, catB3, oxa-10, and aadA1 genes in varying combinations. In addition, heavy metal resistance genes, CopA, merA, and CzcA were positive in 9%, 7%, and 27% of the isolates, respectively. According to these outcomes, the frozen white-leg shrimp in Korean markets can be suggested as a source of multidrug and heavy metal-resistant Aeromonas spp. that carries genetic determinants.

Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland

Distribution of amino acid substitutions in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, parE and determinants of plasmid-mediated quinolone resistance (PMQR) were investigated among quinolone-resistant Salmonella spp. strains isolated from retail food in Poland in the years 2008–2013.Ten different amino acid substitutions were identified in QRDRs. Five different amino acid substitutions were identified in gyrA: Ser83Tyr, Ser83Phe, Asp87Tyr, Asp87Asn, Asp87Gly, two amino acid substitutions in parC: Thr57Ser, Ser80Ile and in parE: Leu445Phe, Arg511Ser. One substitution — Ser464Phe — was detected within gyrB. In gyrA a single substitution (Ser83Tyr) was identified the most frequently – 34.8% (63/181). Second most frequently identified variant (21.0%–38/181) was a co-existence of two single substitutions in gyrA: Ser83Tyr and parC: Thr57Ser. In four isolates co-existed three substitutions in three different genes: gyrA: Ser83Tyr + parC: Thr57Ser + parE: Leu445Phe (two isolates), gyrA: Ser83Phe + parC: Thr57Ser + parE: Leu445Phe, and gyrA: Ser83Tyr + parC: Thr57Ser + parE: Arg511Ser. In the two isolates four substitutions were identified – in gyrA: Ser83Phe + Asp87Tyr and in parC: Thr57Ser + Ser80Ile.Among resistant isolates, MIC values varied between 32 and 2048 mg/L (nalidixic acid) and between 0.125 and 16 mg/L (ciprofloxacin).MIC values of two isolates harboring qnrS1without any substitutions were 32 mg/L (NA) and 0.5–1.0 mg/L (CIP). The highest MIC values for NA and CIP were observed in two isolates of Salmonella spp. carrying double substitutions in gyrA: Ser83Phe + Asp87Tyr and parC: Thr57Ser + Ser80Ile. MIC value for NA was 2048 mg/L while for CIP – 16 mg/L.

Antibiogram of Salmonella Spp Isolates in Kathmandu, Nepal

INTRODUCTION: Drug resistant Salmonella spp. is endemic in several Asian countries. Nalidixic acid-resistant Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A show reduced susceptibility to fluoroquinolones and have resulted in a rise in treatment failures. Over the past few decades, nalidixic acid-resistant Salmonella spp have emerged in Nepal as well.MATERIAL &amp; METHODS: This is a retrospective study that aims to provide a more recent antibiogram of S. Typhi and S. Paratyphi A isolates in Kathmandu. Between Poush, 2071 and Ashwin, 2072 (December 16, 2014 to October 17, 2015), 186 culture positive cases of enteric fever were diagnosed at the Civil Service Hospital. Upon isolation of S. Typhi or S. Paratyphi A, antimicrobial susceptibility testing was performed with amoxicillin, azithromycin, ceftriaxone, chloramphenicol, ciprofloxacin, cotrimoxazole, nalidixic acid, ofloxacin and tetracyciline.RESULTS: This study shows a much higher frequency of nalidixic acid-resistance in Kathmandu than previously reported; 95.7% in Salmonella enterica serovar Paratyphi A and 86.5% in Salmonella enterica serovar Typhi.The rates of ciprofloxacinand ofloxacin-resistance were over 50% in both serovar.CONCLUSION: In Nepal, it is necessary to reevaluate the use of fluoroquinolone therapy and introduce feasible alternatives so as to curb treatment failures.Journal of Universal College of Medical Sciences (2017) Vol. 5, No, 2, Page: 22-25

A Comparative Study Between Cefixime and Ofloxacin in The Treatment of Uncomplicated Typhoid Fever Attending A Tertiary Care Teaching Hospital

Background: Typhoid fever is a gastrointestinal infection caused by Gram-negative bacterium Salmonella enterica serovar typhi (S.typhi). Typhoid fever continues to be one of the major public health problems in developing countries. Antimicrobial therapy is critical for the clinical management of enteric fever. Incidences of multi-drug resistance to S. typhi (MDRST) and nalidixic acid resistant (NAR) strain have limited treatment options. Resistance pattern and time to fever clearance vary in different geographical areas and overtimes. Hence, this study was conducted to compare efficacy and safety profile of Cefixime and Ofloxacin in uncomplicated typhoid fever in this region.Method: 50 adults proven cases of typhoid fever of the age group of 18-57 years of either sex were included in the study. Group I was treated with Cefixime 200 mg twice a day for 7 days and CG I was treated with Ofloxacin 200 mg twice a day for 7 days. Patients were clinically and bacteriologically evaluated during the study period and follow-up.Result: So both study groups were found comparable in terms of mean fever clearance time. 96% cure rates were observed in both groups. No relapse was recorded.Conclusion: Both Cefixime and Ofloxacin are equally efficacious and safe in the treatment of uncomplicated typhoid fever.Med Phoenix Vol.2(1) July 2017, 3-7

Nalidixic acid screening test in detection of decreased fluoroquinolone susceptibility in Salmonella typhi isolated from blood.

To determine the validity of nalidixic acid screening test in the detection of high MICs of fluoroquinolone against Salmonella(S.) typhi isolated from blood and correlate zone diameters of ofloxacin with that of MIC value for nalidixic acid sensitive and resistant strains. Cross-sectional analytical study. Clinical Microbiology Laboratory of the Aga Khan Hospital, Karachi from January 2002 to December 2003. Two hundred S. typhi isolates from blood were included for nalidixic acid screening and ofloxacin susceptibility. Antibiotic susceptibilities for both the antibiotics were obtained by disc diffusion method whereas MICs were determined by standard agar dilution method as recommended by NCCLS guidelines. Sensitivity, specificity and correlation between both antimicrobial susceptibility methods were calculated and results expressed as scattergrams. The results broadly classify S. typhi isolates into nalidixic acid resistant strains with no zone of inhibition around 30 mug nalidixic acid disc and nalidixic acid sensitive strains with mean zone of inhibition of 24.9 mm. All S. typhi isolates with ofloxacin MIC of capital ZHE, Cyrillic 0.125 microg/ml were found to be nalidixic acid resistant (MIC capital ZHE, Cyrillic32 microg/ml) whereas the isolates with ofloxacin MIC 0.06 microg/ml were nalidixic acid sensitive (MIC 8 microg/ml). Screening for nalidixic acid resistance was found to be 100% sensitive and 97% specific in identifying S. typhi strains with reduced susceptibility to fluoroquinolone (MIC capital ZHE, Cyrillic 0.125 microg/ml). Nalidixic acid resistance as a screening method is proved to be significant in identifying S. typhi isolates with reduced susceptibility to fluoroquinolones. It is also suggested that inhibition zone of 25 mm around 5 microg ofloxacin disc is appropriate as a selection criterion to detect S. typhi isolates with reduced susceptibility to fluoroquinolones.

A Randomized Trial to Assess the Effect of Fluoroquinolone Metaphylaxis on the Fecal Prevalence and Quinolone Susceptibilities of Salmonella and Campylobacter in Feedlot Cattle.

The study objective was to determine effects of fluoroquinolone metaphylaxis on fecal prevalence of Salmonella and Campylobacter and fecal prevalence of quinolone-resistant Salmonella and Campylobacter in feedlot cattle. On Day 0, cattle (n = 288) at risk for bovine respiratory disease (BRD) were randomly assigned to either a nontreated control pen (12 pens) or a fluoroquinolone-treated (enrofloxacin; Baytril® 100) pen (12 pens). Rectal fecal samples were collected from cattle on days 0, 7, 14, 21, and 28. Feces were cultured for Salmonella enterica and Campylobacter spp. using enrichment and selective isolation methods, and confirmed by serology and PCR. Susceptibilities to nalidixic acid and ciprofloxacin were determined using microbroth dilution methods. Data analyses were performed using linear mixed models. Overall, Salmonella sp. and Campylobacter spp. were recovered from 10.2% (139/1,364) and 12.4% (170/1,364) of the fecal samples, respectively. Campylobacter species included hyointestinalis, jejuni, and coli. Neither Salmonella sp. nor Campylobacter spp. prevalence was significantly impacted by fluoroquinolone treatment (p = 0.80, p = 0.61, respectively). However, Salmonella prevalence differed between study weeks (p < 0.01) with prevalence decreasing over time. Before treatment, 98.9% (91/92) of Salmonella isolates were susceptible to nalidixic acid and ciprofloxacin. All Salmonella recovered posttreatment (n = 43) were susceptible to both antimicrobials. The majority of Campylobacter spp. recovered before treatment were resistant to nalidixic acid (23/35; 65.7%) and ciprofloxacin (21/35; 60.0%). There was no significant treatment by week interaction (p = 0.85) or treatment effects (p = 0.61) on the posttreatment prevalence of Campylobacter resistance. There was, however, a significant week effect (p = 0.05), with Campylobacter resistance prevalence decreasing over time. In this 28-day study, we found no evidence that a fluoroquinolone used for metaphylaxis significantly impacts fecal prevalence of Salmonella sp. or Campylobacter spp. or the fecal prevalence of nalidixic acid or ciprofloxacin resistance.

The Molecular Mechanisms of Ciprofloxacin Resistance in Clinical Campylobacter jejuni and Their Genotyping Characteristics in Beijing, China.

We assessed the susceptibility of 182 Campylobacter jejuni isolates from patients with diarrhea to eight antibiotics and analyzed the molecular mechanisms of ciprofloxacin resistance as well as the genetic characteristics based on multilocus sequence typing (MLST). The C257T mutation was found on the quinolone resistance-determining region (QRDR) of the gyrA gene in all ciprofloxacin-resistant strains. Mutations on the QRDR of the gyrB gene were silent. A total of 74 strains had 7 inverted repeat (IR) (a 16-bp IR on the intergenic region between cmeR and cmeABC) mutation polymorphisms. Compared with strains without the IR mutations, strains with the IR mutations had higher resistance rates to ciprofloxacin (94.6% vs. 83.3%), nalidixic acid (94.6% vs. 83.3%), tetracycline (98.6% vs. 85.2%), doxycycline (91.9% vs. 71.3%), florfenicol (59.5% vs. 17.6%), chloramphenicol (25.7% vs. 4.6%), gentamicin (16.2% vs. 3.7%), and multidrug resistance than those without IR mutations (all p < 0.05). With C257T mutation alone, 89.9% strains with minimum inhibitory concentration (MIC) values focused on 16, 32, and 64 μg/mL, whereas strains with C257T mutation in combination with the IR mutations had a higher ciprofloxacin resistance level with 88.6% MIC values focused on 64, 128, and 512 μg/mL (p < 0.0001). The strains in this study showed a high genetic variability based on MLST with 117 sequence types (STs), 37 of which were novel. CC-21 was the most common clonal complex (CC) followed by CC-353 and CC-45. No association was found between STs and ciprofloxacin resistance. In conclusion, the C257T mutation on gyrA was the major mechanism for ciprofloxacin resistance, and the C257T mutation in combination with the IR mutations might result in more severe ciprofloxacin resistance to C. jejuni.

Adaptation of Esherichia coli to Antiobiotic Cycling via Single Nucleotide Polymorphisms

Bacteria are quick to adapt and evolve, especially under the effects of selective pressures from chemical antibiotics. In addition, bacteria may develop resistance to antibiotics from multiple classes simultaneously, making their eradication from the human body particularly challenging. This study aims to demonstrate that bacterial multiple-drug resistance can be developed and retained in a laboratory setting. Escherichia coli B was grown in tryptic soy broth in the presence of a small, increasing concentration of streptomycin. This exposure resulted in a strain of E. coli, which had an increased minimum inhibitory concentration (MIC) towards streptomycin, or “resistance.” This resistant strain was then grown in like manner in nalidixic acid and then penicillin G. The result was a strain that became resistant to streptomycin and nalidixic acid, and increasingly resistant to nalidixic acid after penicillin G exposure. Additionally, the bacteria retained resistance to streptomycin and nalidixic acid even after exposure to those chemicals ceased. Genome sequencing and comparison to E. coli B reference strain REL606 revealed the emergence of point mutations with each exposure to an antibiotic. Of particular interest is a mutation associated with the appearance of nalidixic acid resistance. Base pair 4,553,488 was changed from adenine to guanine, resulting in a change from aspartate to glycine in the protein helicase. Previous studies have not indicated mutations to this locus as nalidixic acid resistance conferring. Thus, this mutation may be a novel mutation conferring E. coli B nalidixic acid resistance. Since the region of the mutated helicase is functionally undefined, a mechanism is not apparent. Further research needs to be done to confirm this hypothesis and illuminate a mechanism. KEYWORDS: Bacteria; Escherichia coli; Evolution; Antibiotic Resistance; Nalidixic Acid; Streptomycin; Point Mutation; Single-nucleotide Polymorphism; Helicase; Minimum Inhibitory Concentration

Novel mutations in gyrA and parC among Shigella sonnei strains from Jiangsu Province of China, 2002-2011

BackgroundTo investigate fluoroquinolone resistance and associated mechanisms of Shigella sonnei isolates in Jiangsu Province of China between 2002 and 2011. MethodsAll 337 unduplicated S. sonnei isolates were collected from hospitals in Jiangsu Province from January 2002 to December 2011. Fluoroquinolone susceptibility was characterized by Kirby-Bauer disk diffusion method, and direct nucleotide sequencing of genes of the quinolone resistance determining regions were conducted. Also, the transferable quinolone resistance determinants, including qnrA, qnrB, qnrC, qnrD, qnrS, aac-(6′)-Ib-cr and qepA were amplified by PCR. ResultsAmong 950 Shigella isolates, 337 (35.5%) were identified as S. sonnei, of which 76.6% displayed nalidixic acid resistance and norfloxacin-resistant isolates appeared in 2005-2009, with an average resistance rate of 21.8%. Commonly reported point mutations of Ser83Leu and Asp87Asn/Gly in gyrA and Ser80Ile in parC were detected, with mutation rates of 78.0%, 9.5% and 30.3%, respectively, while no alteration in gyrB or parE were detected. Besides, His211Tyr mutation in gyrA was first reported in a S. sonnei strain in 2009 and two novel mutations in parC were found, of which Met86Trp occurred in another strain in 2009 and Ser129Pro appeared every year except 2011 (28.8%). Plasmid-mediated quinolone resistance determinants were found in 23 isolates and 19 of these isolates were resistant to both nalidixic acid and norfloxacin. qnrB, qnrS, aac-(6′)-Ib-cr and qepA were detected in 1, 7, 14 and 2 S. sonnei strains, relatively, and the most abundant PMQR gene found in this work was aac-(6′)-Ib-cr (4.2%). Interpretation & conclusionsS. sonnei became increasingly important as fluoroquinolone-resistant isolates emerged, and further detection on the resistant genes would be useful in the treatment and control of this infection.

Patterns in antimicrobial susceptibility of Salmonellae isolated at a tertiary care hospital in northern India.

Background & objectives:Multidrug-resistant Salmonellae have emerged worldwide as also in India. The aim of this study was to study the antimicrobial susceptibility pattern of Salmonella enterica serovars isolated at a tertiary care hospital in northern India.Methods:A total of 106 S. enterica serovars isolated from various clinical samples from January 2011 to June 2012 were tested for antimicrobial susceptibility by Kirby-Bauer disk diffusion method. The minimum inhibitory concentration (MIC) of ciprofloxacin, chloramphenicol and ceftriaxone was determined both by agar dilution method and E-test for all the isolates.Results:Salmonella Typhi (73.6%) was the predominant isolate followed by S. Paratyphi A (15.1%), S. Typhimurium (9.4%) and S. Enteritidis (1.9%). Of these, 34 (32.1%) were resistant to ciprofloxacin (MIC ≥1 μg/ml by agar dilution) with MIC90 of ciprofloxacin for S. Typhi, S. Paratyphi A and S. Typhimurium being 32, 4 and 1 μg/ml, respectively. All the isolates were sensitive to chloramphenicol (MIC ≤8 μg/ml) and ceftriaxone (MIC ≤1 μg/ml). Disk diffusion method showed high susceptibility rates to cefotaxime (100%), azithromycin (93.4%) and co-trimoxazole (97.2%). Nalidixic acid resistance was seen in 105 (99.1%) isolates. Of the nalidixic acid-resistant strains, only 34 (32.3%) were found to be resistant to ciprofloxacin (MIC ≥1 μg/ml).Interpretation & conclusions:This study showed an alarming increase in MIC to quinolones and re-emergence of susceptibility to conventional antibiotics among Salmonellae.

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

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

Virulence and Antimicrobial Resistance in Campylobacter spp. from a Peruvian Pediatric Cohort.

The presence of virulence factors (VFs) and mechanisms of quinolones and macrolide resistance was analyzed in Campylobacter spp. from a pediatric cohort study in Lima. In 149 isolates (39 Campylobacter jejuni and 24 Campylobacter coli from diarrheic cases; 57 C. jejuni and 29 C. coli from controls), the presence of the cdtABC and cadF genes and iam marker was established. Nalidixic acid, ciprofloxacin, erythromycin, and azithromycin susceptibilities were established in 115 isolates and tetracycline-susceptibility was established in 100 isolates. The presence of mutations in the gyrA, parC, and 23S rRNA genes was determined. The cadF gene and all genes from the cdtABC operon were significantly more frequent among C. jejuni (P < 0.0001); the iam marker was more frequent in C. coli (P < 0.0001). No differences were observed in VFs between cases and controls. Almost all isolates were tetracycline-resistant; nalidixic acid and ciprofloxacin resistance reached levels of 90.4% and 88.7%, respectively. Resistance to macrolides was 13% (C. jejuni 4.3%; C. coli 26.1%). Resistance to ciprofloxacin was related to GyrA Thr86 substitutions, while 13 of 15 macrolide-resistant isolates possessed a 23S rRNA mutation (A2075G). Differences in the presence of VFs and alarming levels of resistance to tested antimicrobial agents were observed among C. jejuni and C. coli.