Tea polyphenols and catechins postpone evolution of antibiotic resistance genes and alter microbial community under stress of tetracycline.

Relying on the high mobility of water flow, the dissemination of antibiotic resistance genes (ARGs) in the water tends to be exacerbated and enlarged. It caused negative impacts on a wider scope of the environment. The ARGs dissemination monitoring and the methods efficiently reducing their concentration in water became the focus of interest. Green chemicals with antibacterial effects such as tea polyphenols (TPs) and catechins (CA) have been considered as auxiliary disinfectants for ARGs removal in the water environment. However, the antibacterial performance of TPs and CA under the stress of external antibiotics still lacks sufficient research. The results show that more operational taxonomic units can be observed in water samples with TPs and CA than in those without the ingredients under pressure of tetracycline. An unexpected increase along with the increase of ARGs concentrations and the diversity of microbial communities under the low-concentration TPs or CA (1 mg/L). Besides, under the stress of tetracycline, the inhibition of TPs was detected to be strengthened for increase of inti1 and tetC but weakened towards for the increase of tetA. Whilst CA substantially diminished abundances of tetC and tetA under tetracycline pressure. This research demonstrated that TPs and CA are able to assuage development of ARGs under the pressure of antibiotic in water system.

Plasmid mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.

Antimicrobial resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus CC398 isolates from Spanish hospitals.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of lineage CC398 is an emerging clone causing human infections but is mostly found in pigs. The aim of this study was to characterize the antimicrobial resistance phenotypes/genotypes of a collection of 137 MRSA CC398 isolates obtained in a previous study from 17 Spanish hospitals, using tetracycline resistance as marker for selection. A multidrug-resistant (MDR) phenotype was present in 79% of analysed isolates, with 17% of them resistant to at least six different antimicrobial families. All tetracycline-resistant isolates (n=137) carried the tetM gene and 75% also carried the tetK gene. Almost 50% of MRSA CC398 isolates showed macrolide and/or lincosamide resistance: a) 39% of isolates were ERYR-CLIR (all with constitutive phenotype), with 87% of them carrying the ermC gene, followed by msrA (25%), ermB (21%), vgaA (17%), ermA (6%), lsaB (4%), linA (2%), linB (2%), and ermT (2%, this isolate with the new spa-type t18071); and b) 9% of MRSA CC398 isolates showed the dissociated ERYS-CLIR phenotype carrying the linA, linB, lsaB and vgaA genes. Other antimicrobial resistance phenotypes in these MRSA CC398 isolates included resistance to ciprofloxacin (67%), aminoglycosides (21%), mupirocin (6%), chloramphenicol (4%) or fusidic acid (2%). The more common resistance genes detected for some of these antimicrobials were: aac(6')-Ie-aph(2'')-Ia (16%) and ant(4')-Ia (12%) for aminoglycosides, and fexA (3%) for chloramphenicol. The high rate of MDR phenotypes with a wide range of antimicrobial resistance genes shown in this study reduce the potential therapeutic options in case of infections.

Roles of ramR and tet(A) Mutations in Conferring Tigecycline Resistance in Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates.

Tigecycline is regarded as a last-resort treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, but increasing numbers of tigecycline-resistant K. pneumoniae isolates have been reported. The tigecycline resistance mechanisms in CRKP are undetermined. This study aimed to elucidate the mechanisms underlying tigecycline resistance in 16 tigecycline- and carbapenem-resistant K. pneumoniae (TCRKP) isolates. Mutations in tigecycline resistance determinant genes [ramR, acrR, oqxR, tet(A), tet(L), tet(X), tet(M), rpsJ] were assessed by PCR amplicon sequencing, and mutations in ramR and tet(A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional ramR mutation profiles reducing tigecycline sensitivity were verified by plasmid complementation of wild-type and mutant ramR Using a site-specific mutant, the most frequent RamR mutation, A19V (60%), had no significant effect on tigecycline susceptibility or the upregulation of ramA and acrA Two tet(A) variants with double frameshift mutations, type 1 and type 2, were identified; type 2 tet(A) is novel. A parent strain transformed with a plasmid carrying type 1 or type 2 tet(A) increased the tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring no ramR gene and a mutated tet(A), with an 8-fold increase in the tigecycline MIC compared with that in strains harboring only mutated tet(A) being seen. Overall, mutations in the ramR and tet(A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of a ramR deficiency and widespread mutated tet(A) is concerning due to the possible dissemination of increased tigecycline resistance in K. pneumoniae.

The Chlamydia suis Genome Exhibits High Levels of Diversity, Plasticity, and Mobile Antibiotic Resistance: Comparative Genomics of a Recent Livestock Cohort Shows Influence of Treatment Regimes.

Chlamydia suis is an endemic pig pathogen, belonging to a fascinating genus of obligate intracellular pathogens. Of particular interest, this is the only chlamydial species to have naturally acquired genes encoding for tetracycline resistance. To date, the distribution and mobility of the Tet-island are not well understood. Our study focused on whole genome sequencing of 29 C. suis isolates from a recent porcine cohort within Switzerland, combined with data from USA tetracycline-resistant isolates. Our findings show that the genome of C. suis is very plastic, with unprecedented diversity, highly affected by recombination and plasmid exchange. A large diversity of isolates circulates within Europe, even within individual Swiss farms, suggesting that C. suis originated around Europe. New World isolates have more restricted diversity and appear to derive from European isolates, indicating that historical strain transfers to the United States have occurred. The architecture of the Tet-island is variable, but the tetA(C) gene is always intact, and recombination has been a major factor in its transmission within C. suis. Selective pressure from tetracycline use within pigs leads to a higher number of Tet-island carrying isolates, which appear to be lost in the absence of such pressure, whereas the loss or gain of the Tet-island from individual strains is not observed. The Tet-island appears to be a recent import into the genome of C. suis, with a possible American origin.

Autoclave treatment of pig manure does not reduce the risk of transmission and transfer of tetracycline resistance genes in soil: successive determinations with soil column experiments.

The increasing use of antibiotics, especially tetracycline, in livestock feed adversely affects animal health and ecological integrity. Therefore, approaches to decrease this risk are urgently needed. High temperatures facilitate antibiotic degradation; whether this reduces transmission risk and transfer of tetracycline-resistant bacteria (TRBs) and tetracycline resistance genes (TRGs) in soil remains unknown. Successive experiments with soil columns evaluated the effects of autoclaving pig manure (APM) on soil TRB populations and TRGs over time at different soil depths. The data showed sharp increases in TRB populations and TRGs in each subsoil layer of PM (non-APM) and APM treatments within 30 days, indicating that TRBs and TRGs transferred rapidly. The level of TRBs in the upper soil layers was approximately 15-fold higher than in subsoils. TRBs were not dependent on PM and APM levels, especially in the late phase. Nevertheless, higher levels of APM led to rapid expansion of TRBs as compared to PM. Moreover, temporal changes in TRB frequencies in total culturable bacteria (TCBs) were similar to TRBs, indicating that the impact of PM or APM on TRBs was more obvious than for TCBs. TRBs were hypothesized to depend on the numbers of TRGs and indigenous recipient bacteria. In the plough layer, five TRGs (tetB, tetG, tetM, tetW, and tetB/P) existed in each treatment within 150 days. Selective pressure of TC may not be a necessary condition for the transfer and persistence of TRGs in soil. High temperatures might reduce TRBs in PM, which had minimal impact on the transmission and transfer of TRGs in soil. Identifying alternatives to decrease TRG transmission remains a major challenge.

Emergence and spread of plasmid-borne tet(B)::ISCR2 in minocycline-resistant Acinetobacter baumannii isolates.

Resistance to minocycline has emerged in multidrug-resistant Acinetobacter baumannii isolates from Buenos Aires hospitals. Few reports about the description and dispersion of tet genes in this species have been published. We observed the presence of tet(B) in all minocycline-resistant isolates. This gene was found to be associated with the ISCR2 mobile element, which may, in part, explain its dispersion.

Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand.

AIMS: This study aimed at surveying prevalence of oxytetracycline (OTC)-resistant bacteria in the white-leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC-resistant genes and multiple-antibiotic resistance in the isolates. METHODS AND RESULTS: Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC-resistant bacteria were 0·3-52·1% in white-leg samples and 0·008-22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC-resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4-128 µg ml(-1) in the OTC-resistant aeromonads and 128-256 µg ml(-1) in OTC-resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. CONCLUSIONS: Both species of shrimp are associated with OTC-resistant bacteria, occasionally at high densities exceeding 10(6) cfu g(-1). The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC-resistant genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Cultured shrimps can be vehicle to carry OTC-resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC-resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.

Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline-amended and ciprofloxacin-amended growth media.

AIMS: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. METHODS AND RESULTS: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline- (n=164) and ciprofloxacin-amended (n=65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin-amended growth media (62%) compared to the bacteria isolated on tetracycline-amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline-resistant bacteria and almost half of the ciprofloxacin-resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline-resistant bacteria was capable of lateral gene transfer. CONCLUSIONS: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline-resistant and ciprofloxacin-resistant bacteria in municipal wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.

Longitudinal characterization of resistant Escherichia coli in fecal deposits from cattle fed subtherapeutic levels of antimicrobials.

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp(r)) and tetracycline-resistant (Tet(r)) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp(r) and Tet(r) E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet(r) E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet(r) and Amp(r) E. coli. Except for Amp(r) E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet(r) E. coli numbers were detected in A44 and AS700 deposits [5.9 log(10) CFU (g dry matter)(-1) and 5.4 log(10) CFU (g dry matter)(-1), respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla(TEM1), tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.

Tetracycline resistance genes and tetracycline resistant lactose-fermenting Enterobacteriaceae in activated sludge of sewage treatment plants.

Activated sludges were sampled from five sewage treatment plants (STPs) distributed in three geographically isolated areas, i.e., Hong Kong (Shatin, Stanley), Shanghai (Minhang) in China, and the bay area in California (Palo Alto and San Jose) of the United States. Among the tested 14 tetracycline resistance (tet) genes, nine genes encompassing efflux pumps (tetA, tetC, tetE, and tetG), ribosomal protection proteins (tetM, tetO, tetQ, and tetS), and enzymatic modification (tetX) were commonly detected in the STP sludge samples, whereas five genes encompassing efflux pumps [tetB, tetD, tetL, tetK, and tetA(P)] were not detected in any sludge sample. Additionally, 109 lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from the activated sludge of the Shatin STP. Tetracycline-resistant (TR) LFE accounted for 32% of the total 109 LFE strains. The occurrence frequencies of tet genes among all TR-LEF strains varied from 0 to 91%, i.e., tetC (91%), tetA (46%), tetE (9%), tetG (6%), and tetD (6%). Finally, quantitative real-time polymerase chain reaction was used to quantify the change of tetC and tetA genes as the indicator of TR-LEF in the Shatin and Stanley STPs. The results showed that the concentrations of tetC and tetA genes in STP effluent ranged from 10(4) to 10(5) copies/mL, significantly lower than those in the influent by 3 orders of magnitude.

Fate of tetracycline resistance genes in aquatic systems: migration from the water column to peripheral biofilms.

Antibiotic resistance genes (ARGs) are emerging contaminants that are being found at elevated levels in sediments and other aquatic compartments in areas of intensive agricultural and urban activity. However, little quantitative data exist on the migration and attenuation of ARGs in natural ecosystems, which is central to predicting their fate after release into receiving waters. Here we examined the fate of tetracycline-resistance genes in bacterial hosts released in cattle feedlot wastewater using field-scale mesocosms to quantify ARG attenuation rate in the water column and also the migration of ARGs into peripheral biofilms. Feedlot wastewater was added to fifteen cylindrical 11.3-m3 mesocosms (some of which had artificial substrates) simulating five different receiving water conditions (in triplicate), and the abundance of six resistance genes (tet(O), tet(W), tet(M), tet(Q), tet(B), and tet(L)) and 16S-rRNA genes was monitored for 14 days. Mesocosm treatments were varied according to light supply, microbial supplements (via river water additions), and oxytetracycline (OTC) level. First-order water column disappearance coefficients (kd) for the sum of the six genes (tetR) were always higher in sunlight than in the dark (-0.72 d(-1) and -0.51 d(-1), respectively). However, water column kd varied among genes (tet(O) < tet(W) < tet(M) < tet(Q); tet(B) and tet(L) were below detection) and some genes, particularly tet(W), readily migrated into biofilms, suggesting that different genes be considered separately and peripheral compartments be included in future fate models. This work provides the first quantitative field data for modeling ARG fate in aquatic systems.

Influence of oral doxycycline therapy on the diversity and antibiotic susceptibility of human intestinal bifidobacterial population.

AIM: To evaluate the influence of doxycycline therapy on the composition and antibiotic susceptibility of intestinal bifidobacteria. METHODS AND RESULTS: Faecal samples were collected from nine subjects receiving doxycycline therapy and ten control subjects, and analysed for bifidobacteria by culturing and PCR-DGGE (denaturing gradient gel electrophoresis). A marked decrease in the diversity (average number of amplicons detected by PCR-DGGE 0.8 in the antibiotic vs 4.3 in the control group) of Bifidobacterium populations was observed during doxycycline therapy. The proportion of a tetracycline-resistant bifidobacterial population was higher in the antibiotic group than in the control group (83%vs 26%). Based on the tet gene PCR, resistance could be associated with the presence of tet(W). In two subjects, strains representing highly similar genetic fingerprints but different tetracycline susceptibilities were detected. A mutation causing lack of functionality in the tet(W) was observed in one of the susceptible strains. CONCLUSIONS: Doxycycline therapy had a drastic effect on the diversity and tetracycline susceptibility of intestinal Bifidobacterium populations. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of broad-spectrum antibiotics increased the pool of tetracycline-resistant commensal bacteria in the intestine. The detection of resistance genes alone is not sufficient for the evaluation of bacterial antibiotic resistance.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5 percent of the samples were positive for tetA, and also 37.5 percent were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

Effect of tetracycline residues in pig manure slurry on tetracycline-resistant bacteria and resistance gene tet(M) in soil microcosms.

Effects of tetracycline residues from pig manure slurry on the prevalence of tetracycline-resistant bacteria and the tetracycline resistance gene, tet(M), were studied in soil microcosms. Four types of soil microcosms were established for a period of 152 days, supplemented with combinations of pig manure slurry and a tetracycline-resistant Enterococcus faecalis, CG110, containing the tetracycline resistance gene tet(M) (on the conjugative transposon, Tn916). The prevalence of both tetracycline-resistant aerobic bacteria and tetracycline-resistant enterococci declined rapidly until day 45 where no significant differences in the levels of tetracycline-resistant bacteria in any of the four types of microcosms could be detected. tet(M) could be detected in microcosms supplemented with either pig manure slurry and/or E. faecalis CG110 (tet(M)) for the whole period (152 days). tet(M) could be detected longer than tetracycline-resistant enterococci could be isolated (limit of detection 100 CFU/g soil) probably due to viable but not culturable (VBNC) bacteria with tet(M), horizontal gene transfer of tet(M) to indigenous soil bacteria or presence of "free" DNA. The concentration of chlortetracycline and oxytetracycline were almost stable through out the experimental period, but the tetracycline concentrations had no effect on prevalence of tetracycline-resistant bacteria. The presented microcosm approach simulated natural farmland conditions well and supported results from previous field studies.

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig.

AIMS: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracycline-resistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. METHODS AND RESULTS: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l-1 and a 10% increase in E. coli with an MIC > 50 mg l-1 during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 microg g-1 faeces during treatment. CONCLUSION: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.

Detection of tetracyclines and efflux pump inhibitors.

Screening assays for the detection of tetracyclines and inhibitors of tetracycline efflux pumps are described. The tetracycline assay is based on the observation that the tetA(B) gene encoding the efflux pump of transposon Tn10 is induced by tetracycline. The Escherichia coli strain designed to detect tetracyclines contains a single copy of a tetA(B)-lacZ transcriptional fusion integrated into the chromosome and the tetR gene encoding the tetracycline repressor on a plasmid. The assay specifically detects tetracyclines of distinct structures, but not other classes of drugs. A strain capable of detecting inhibitors of the TetA(B) efflux pump contained the tetA(B)-lacZ fusion and, in addition, a tetA(B) structural gene lacking its transcriptional regulatory signals which mediated resistance to only 5 micrograms of tetracycline per ml. This strain was more refractory to induction by tetracycline because of the action of the pump. Inhibitors were detected in two ways: (i) beta-galactosidase induction in the presence of 5 ng of tetracycline per ml, a subinducing concentration, and (ii) growth inhibition in the presence of 5 micrograms of tetracycline per ml. A strain designed to detect inhibitors of the Tet(K) efflux pump from Staphylococcus aureus was constructed by substituting the tet(K) structural gene for the tetA(B) gene. Nocardamine and other siderophores were found to interfere with the action of tetracycline efflux pumps.