A laboratory-based survey of Campylobacter infections in Prahova County

In developing countries, as well as in many western countries, members of the genus Campylobacter are recognized as one of the most common cause of acute bacterial enteritis. Campylobacter jejuni and Campylobacter coli isolation rates have been shown to be equal to, and sometimes higher than those of other enteric pathogens. The Microbiology Laboratory of the local Public Health Authority in Prahova County conducted a one and a half-year laboratory-based survey of Campylobacter infections in patients suffering from gastrointestinal symptoms. From a total of 3284 stool samples screened, the culture-positive ones confirmed the bacterial etiology for 551 diarrhea cases. Campylobacter was found in 345 specimens, being the most frequently isolated enteropathogen. C. jejuni outnumbered C. coli species (239 vs. 106 isolates). Salmonella isolates were the second local cause of diarrhea. The highest isolation rate of Campylobacter was found in children 5 years of age (262 strains). The prevalence of campylobacteriosis declined with age. The isolation rate of Campylobacter (10.5%), the unimodal age-specific distribution of cases, as well as the identification of polymicrobial infections among the screened population were epidemiological aspects resembling reports on campylobacteriosis in developing countries. The susceptibility of Campylobacter isolates to various antimicrobial agents, including macrolides and fluoroquinolones was also assessed. Among the screened isolates, Erythromycin retained a good activity, while an increased ciprofloxacin resistance was observed. The information gathered through this local study sustains the importance of Campylobacter in the etiology of autochthonous infectious diarrhea. A development of a national surveillance program regarding the most important foodborne pathogens would be beneficial for improving prevention and controlling measures.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni and Campylobacter coli are recognized as the most common causative agents of bacterial gastroenteritis in the world and infections with these organisms occur more frequently than do infections due to Salmonella species, Shigella species, or Escherichia coli 0157:H7. The incidence of human Campylobacter infections has increased markedly in both developed and developing countries worldwide and, more significantly, so has the rapid emergence of antibiotic-resistant Campylobacter strains, with evidence suggesting that the use of antibiotics, in particular the fluoroquinolones, as growth promoters in food animals and the veterinary industry is accelerating this trend. In this minireview, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter spp are discussed.     

Temporal prevalence of antimicrobial resistance in Campylobacter spp. from beef cattle in Alberta feedlots

Antimicrobial resistance (AMR) was temporally assessed in campylobacters isolated from beef cattle (7,738 fecal samples from 2,622 animals) in four commercial feedlots in Alberta. All calves were administered chlortetracycline and oxytetracycline in feed, and a majority of the animals (93%) were injected with long-acting oxytetracycline upon arrival at the feedlot. Fecal samples from individual animals were collected upon arrival (i.e., entry sample), 69 days (standard deviation [SD] = 3 days) after arrival (i.e., interim sample), and 189 days (SD = 33 days) after arrival (i.e., exit sample) at the feedlot. In total, 1,586 Campylobacter isolates consisting of Campylobacter coli (n = 154), Campylobacter fetus (n = 994), Campylobacter jejuni (n = 431), Campylobacter hyointestinalis (n = 4), and Campylobacter lanienae (n = 3) were recovered and characterized. The administration of antimicrobials did not decrease carriage rates of campylobacters, and minimal resistance (< or =4%) to azithromycin, ciprofloxacin, enrofloxacin, gentamicin, and meropenem was observed. In contrast, substantive increases in the prevalence of isolates resistant to tetracycline and doxycycline (56 to 89%) for C. coli, C. fetus, and C. jejuni, as well as in the number of animals (7 to 42%) from which resistant isolates were recovered, were observed during the feedlot period. Increased resistance to erythromycin (total isolates and carriages rates) was also observed in isolates of C. coli over the three isolation times. The majority of C. fetus isolates recovered were resistant to nalidixic acid, but this was independent of when they were isolated. A relatively limited number of multidrug-resistant isolates were recovered and consisted primarily of C. coli resistant to tetracyclines and erythromycin (10% of isolates). Over the course of the feedlot period, considerable increases in antimicrobial resistance were observed in C. coli, C. fetus, and C. jejuni, but with the exception of erythromycin resistance in C. coli, the administration of antimicrobial agents to beef cattle was found to have a minimal impact on resistance to macrolides and fluoroquinolones, the two classes of antimicrobials used to treat campylobacteriosis in humans. However, the widespread use of antimicrobial agents in beef production and the possible horizontal transfer of mobile genetic elements with antimicrobial resistance determinants among Campylobacter and other bacterial taxa emphasize the need to monitor AMR development in bacteria from beef cattle.     

Efflux-mediated drug resistance in Gram-positive bacteria

Gram-positive bacteria express numerous membrane transporters that promote the efflux of various drugs, including many antibiotics, from the cell to the outer medium. Drug transporters can be specific to a particular drug, or can have broad specificity, as in so-called multidrug transporters. This broad specificity can be a consequence of the hydrophobic nature of transported molecules, as suggested by recent structural studies of soluble multidrug-binding proteins. Although the functions of drug transporters may involve both the protection of bacteria from outside toxins and the transport of natural metabolites, their clinical importance lies largely in providing Gram-positive pathogens with resistance to macrolides, tetracyclines and fluoroquinolones. A number of agents, discovered in recent years, that inhibit drug transporters can potentially be used to overcome efflux-associated antibiotic resistance.     

Antimicrobial resistance in Campylobacter: Susceptibility testing methods and resistance trends

Most Campylobacter infections are self-limiting but antimicrobial treatment (e.g., macrolides, fluoroquinolones) is necessary in severe or prolonged cases. Susceptibility testing continues to play a critical role in guiding therapy and epidemiological monitoring of resistance. The methods of choice for Campylobacter recommended by the Clinical and Laboratory Standards Institute (CLSI) are agar dilution and broth microdilution, while a disk diffusion method was recently standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Macrolides, quinolones, and tetracyclines are among the common antimicrobials recommended for testing. Molecular determination of Campylobacter resistance via DNA sequencing or PCR-based methods has been performed. High levels of resistance to tetracycline and ciprofloxacin are frequently reported by many national surveillance programs, but resistance to erythromycin and gentamicin in Campylobacter jejuni remains low. Nonetheless, variations in susceptibility observed over time underscore the need for continued public health monitoring of Campylobacter resistance from humans, animals, and food.     

Prevalence and antimicrobial resistance of Campylobacter in US dairy cattle

AIMS: To obtain an overview of the prevalence and antimicrobial resistance of Campylobacter in faeces of US dairy cows in 2002. METHODS AND RESULTS: Faeces from 1435 cows, representing 96 dairy operations in 21 US states, were collected for the culture of Campylobacter. A total of 735 Campylobacter strains were isolated (51.2% positive samples) with 94 operations positive (97.9%) for Campylobacter. From this collection, 532 isolates (473 Campylobacter jejuni and 59 Campylobacter coli) were randomly selected for susceptibility testing to eight antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. The C. jejuni isolates exhibited resistance to tetracycline (47.4%), nalidixic acid (4.0%) and ciprofloxacin (2.5%), while the C. coli strains exhibited some resistance to all antimicrobials except chloramphenicol and ciprofloxacin. Only 3.6% of the C. jejuni isolates were resistant to two or more antimicrobials but 20.3% of the C. coli strains were multiresistant. CONCLUSIONS: On most operations, at least one cow was positive for Campylobacter and more than half of the cows sampled were shedding Campylobacter. The C. coli isolates had significantly higher levels of resistance to macrolides and to tetracycline compared with the C. jejuni strains, but were susceptible to ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a high prevalence of Campylobacter on US dairy operations; however, US dairy cattle have not been recognized as a major source of human infection compared with poultry. Campylobacter coli appears to develop antimicrobial resistance more readily than C. jejuni from the same environment.     

Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

The epidemiology of antibiotic resistance in Campylobacter

Antibiotic resistance, particularly with the fluoroquinolones and macrolide antibiotics, has now emerged globally with thermophilic campylobacters, including Campylobacter jejuni and C. coli, giving rise to concerns about how these organisms have acquired such resistance characteristics, as well as consequences for human and animal treatment. This review examines (i) the clinical epidemiology of antibiotic resistance in human and animal thermophilic campylobacters, (ii) an update on resistance rates globally, (iii) surveillance of antimicrobial resistance in campylobacters originating from animals, particularly poultry, (iv) the role of the environment in the acquisition and transmission of antibiotic-resistant campylobacters, as well as (v) issues of biocide resistance in campylobacters.     

The solithromycin journey—It is all in the chemistry

The macrolide class of antibiotics, including the early generation macrolides erythromycin, clarithromycin and azithromycin, have been used broadly for treatment of respiratory tract infections. An increase of treatment failures of early generation macrolides is due to the upturn in bacterial macrolide resistance to 48% in the US and over 80% in Asian countries and has led to the use of alternate therapies, such as fluoroquinolones. The safety of the fluoroquinolones is now in question and alternate antibiotics for the outpatient treatment of community acquired bacterial pneumonia are needed. Telithromycin, approved in 2003, is no longer used owing to serious adverse events, collectively called the ‘Ketek effects’. Telithromycin has a side chain pyridine moiety that blocks nicotinic acetylcholine receptors. Blockade of these receptors is known experimentally to cause the side effects seen with telithromycin in patients use. Solithromycin is a new macrolide, the first fluoroketolide, which has been tested successfully in two Phase 3 trials and is undergoing regulatory review at the FDA. Solithromycin is differentiated from telithromycin chemically and biologically in that its side chain is chemically different and does not significantly block nicotinic acetylcholine receptors. Solithromycin was well tolerated and effective in clinical trials.     

Chemotherapy of Campylobacter infections]

A total of 600 patients with suspected alimentary food poisoning were hospitalized. The ++clinico-laboratory findings showed that 27 (4.5 per cent) of them had Campylobacter infection. The cultures of Campylobacter jejuni and Campylobacter coli were isolated from 24 and 3 patients, respectively. The patients underwent complex pathogenetic treatment with oral rehydration saline solutions, symptomatic agents, enzymatic preparations and diet (the basic therapy) supplemented with biological bacterial preparations in less severe cases (8 patients). Antibacterial drugs such as furazolidone (roxytromycin), gentamicin and levomycetin, as well as fluoroquinolones such as ofloxacin and ciprofloxacin were additionally used in the treatment of both the patients with the generalized infection and those with more severe processes of the disease, pronounced diarrhea with blood traces and persisting isolation of Campylobacter (12 patients). The most favourable results in the treatment of more severe patients with Campylobacter infection were obtained with the fluoroquinolones used after inadequately efficient therapy with furazolidone or antibiotics especially in the cases with repeated isolation of campylobacteria.     

Genomic analysis of the diversity,antimicrobial resistance and virulence potential of clinical Campylobacter jejuni and Campylobacter coli strains from Chile

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n = 81), collected in 2017–2019 in Santiago, Chile. This culture collection accounts for more than one third of the available genome sequences from South American clinical strains. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen.     

Membrane interactions of non-membrane targeting antibiotics: The case of aminoglycosides,macrolides, and fluoroquinolones

Numerous antibiotics are known to target intracellular pathways, such as protein translation or DNA replication. Membrane transporters typically regulate drug uptake; however, little is known about direct interactions between these antibiotics and the cell membranes. Here, we studied the interactions between different aminoglycosides (kanamycin, gentamicin, streptomycin, neomycin), macrolides (azithromycin, clarithromycin, erythromycin), and fluoroquinolones (ciprofloxacin, levofloxacin) with bacterial membrane mimics to determine drug partitioning and potential drug-induced membrane disruption. The antibiotics' exact location in the bilayers and their effect on membrane thickness and fluidity were determined from high-resolution X-ray diffraction. While the antibiotics did not change membrane thickness at low (1:100 drug/lipid) or high (1:10 drug/lipid) concentrations, they were found to increase membrane disorder in a dose-dependent manner. However, no membrane damage, such as membrane disruption or pore formation, was observed for any of the antibiotics. To note, all antibiotics partitioned into the lipid head groups, while macrolides and fluoroquinolones also partitioned into the bilayer core. The results suggest that the bacterial membrane is relatively inert in the direct mechanisms of actions of these antibiotics.     

The ability of certain antimutagenic agents to prevent development of antibiotic resistance

Resistance to multiple antimicrobial agents has now become a prominent fact of contemporary life. It is believed that poor patient compliance, e.g. interrupted or premature cessation of therapy; and misuse or abuse of antibiotics, e.g. wrong antibiotic or insufficient dose, play important roles in resistance development. We present evidence that, this form of resistance often stems from spontaneous mutations accompanied by the positive selecting pressure of the doses of antibiotics being between the MIC and MBC levels. A number of antimutagenic agents, e.g. green tea catechins, and other antioxidants, etc. are able to suppress the emergence of resistance. In many cases, these agents are capable of exerting these effects at doses which by themselves produce no visible effect on growth. In a number of cases antimutagenic substances capable of preventing resistance emergence are present in normal food stuffs. These effects are exerted against resistance to tetracyclines, fluoroquinolones, macrolides, beta-lactams, aminoglycosides and the like. The implications of these laboratory findings for practical chemotherapy are discussed.     

Tigecicline susceptibilities of tetracycline-resistant Campylobacter jejuni clinical strains isolated in Poland

Campylobacteriosis is a significant public health problem in many developed countries. Campylobacter jejuni is one of the leading causes of food-borne gastroenteritis and enteritis in humans. Treatment of campylobacteriosis is required in severe clinical infections, extraintestinal infections and in immunocompromised patients. Erythromycin is the proposed drug of choice for the treatment of Campylobacter infections. However, tetracycline and fluoroquinolones (ciprofloxacin) are also clinically effective agents for treating infections caused by Campylobacter spp. High prevalence of C. jejuni resistant to fluoroquinolone and tetracycline have been recently reported in many countries. In human medicine new agents for the treatment of many serious infections are acutely needed in hospital practice. Tigecycline is a member of a new group of antibiotics--the glycylcyclines with an expanded microbiological spectrum. In our study we will determine the susceptibility of polish, resistant to tetracycline clinical C. jejuni isolates to tigecycline. All 94 tetracycline-resistant C. jejuni strains, with MICs between 8 and 256 mg/l, isolated between 2007 and 2008 were susceptible to tigecycline, with MICs 0.06 mg/1. Tigecycline may has potential therapeutic role in the treatment of serious Campylobacter infections.     

Etiology of severe hospital infections in intensive care units and antibiotic resistance of pathogens]

The leading pathogens of severe infections in intensive care units were the following: respiratory tract infections--bacteria of the famility of Enterobacteriaceae (33.8%), Pseudomonas spp. (24.9%), Acinetobacter spp. (18.1%), Staphylococcus aureus (16.0%), blood flow infections--coagulase negative staphylococci (33.6%), S. aureus (26.1%), Enterobacteriaceae (17.6%), wound infections--Enterobacteriaceae (35.7%), coagulase negative staphyloccocci (17.8%), Pseudomonas spp. (14.3%). As for various species of Enterobacteriaceae, susceptibility was preserved in 91-100% of the isolates to meropenem, in 72-100% to cefoperazone/sulbactam, in 51-65% to cefepime, in 72-86% to amikacin, and in less than 50% to cephalosporins and fluoroquinolones. As for P.aeruginosa, 28% of the isolates was resistant to all the antibacterials, except polymyxin. The highest susceptibility to cefoperazone/sulbactam and meropenem was revealed in the isolates of Acinetobacter baumannii. Oxacillin resistance was detected in 64.9% of the S.aureus isolates. The oxacillin resistance as a rule was associated with resistance to macrolides, aminoglycosides and fluoroquinolones. As for coagulase negative staphylococci, oxacillin resistance was stated in 75.6% of the isolates. All the isolates of the Staphylococcus spp. preserved their susceptibility to vancomycin and linezolid.     

Identification and molecular characterisation of CmeB, a Campylobacter jejuni multidrug efflux pump

A multidrug efflux pump gene (cmeB) was identified from the published Campylobacter jejuni genome sequence. Secondary structural analysis showed that the gene encoded a protein belonging to the resistance nodulation cell division (RND) family of efflux transporters. The gene was inactivated by insertional mutagenesis. Compared with the wild-type strain (NCTC 11168), the resultant knockout strain (NCTC 11168-cmeB::kan(r)) displayed increased susceptibility to a range of antibiotics including beta-lactams, fluoroquinolones, macrolides, chloramphenicol, tetracycline, ethidium bromide, the dye acridine orange and the detergent sodium dodecyl sulfate. Accumulation of ciprofloxacin was increased in the knockout mutant, but carbonyl cyanide m-chlorophenyl hydrazone, a proton motive force inhibitor, had less effect upon ciprofloxacin accumulation in the knockout mutant compared with NCTC 11168. These data show that the identified gene encodes an RND-type multi-substrate efflux transporter, which contributes to intrinsic resistance to a range of structurally unrelated compounds in C. jejuni. This efflux pump has been named CmeB (for Campylobacter multidrug efflux).     

Antimicrobial resistance in Campylobacter isolated from food animals and humans in northern Thailand

A study was conducted to determine the prevalence of Campylobacter with antimicrobial resistance from chickens, pigs, dairy cows, healthy farm workers, and children hospitalized with diarrhea in northern Thailand. Resistance was highest in pig samples and lowest in healthy farm workers. Resistance to fluoroquinolones and tetracycline was high in all study populations. The increased prevalence of resistant isolates from meat samples collected at markets, compared to isolates collected from animals on the farm or the slaughterhouse, suggests that contamination after carcasses leave the slaughterhouse is an important factor in the spread of resistant bacteria into the human food chain.     

Exploring the permeation of fluoroquinolone metalloantibiotics across outer membrane porins by combining molecular dynamics simulations and a porin-mimetic in vitro model

The misuse and overuse of fluoroquinolones in recent years have triggered alarming levels of resistance to these antibiotics. Porin channels are crucial for the permeation of fluoroquinolones across the outer membrane of Gram-negative bacteria and modifications in porin expression are an important mechanism of bacterial resistance. One possible strategy to overcome this problem is the development of ternary copper complexes with fluoroquinolones. Compared to fluoroquinolones, these metalloantibiotics present a larger partition to the lipid bilayer and a more favorable permeation, by passive diffusion, across bacteriomimetic phospholipid-based model membranes. To rule out the porin-dependent pathway for the metalloantibiotics, we explored the permeation through OmpF (one of the most abundant porins present in the outer membrane of Gram-negative bacteria) using a multi-component approach. X-ray studies of OmpF porin crystals soaked with a ciprofloxacin ternary copper complex did not show a well-defined binding site for the compound. Molecular dynamics simulations showed that the translocation of the metalloantibiotic through this porin is less favorable than that of free fluoroquinolone, as it presented a much larger free energy barrier to cross the narrow constriction region of the pore. Lastly, permeability studies of different fluoroquinolones and their respective copper complexes using a porin-mimetic in vitro model corroborated the lower rate of permeation for the metalloantibiotics relative to the free antibiotics. Our results support a porin-independent mechanism for the influx of the metalloantibiotics into the bacterial cell. This finding brings additional support to the potential application of these metalloantibiotics in the fight against resistant infections and as an alternative to fluoroquinolones.